fmrfamide and proctolin

Technological advancements in high-throughput sequencing have resulted in the production/public deposition of an ever-growing number of arthropod transcriptomes. While most sequencing projects have focused on hexapods, transcriptomes have also been generated for members of the Chelicerata. One chelicerate for which a large transcriptome has recently been released is the Western black widow Latrodectus hesperus, a member of the Araneae (true spiders). Here, a neuropeptidome for L. hesperus was predicted using this resource. Thirty-eight peptide-encoding transcripts were mined from the L. hesperus transcriptome, with 216 distinct peptides predicted from the deduced pre/preprohormones. The identified peptides included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon β, CAPA/periviscerokinin/pyrokinin, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, neuropeptide F (NPF), orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. Of particular note were the identifications of a carboxyl (C)-terminally extended corazonin, FLPs possessing -IMRFamide, -MMYFamide, and -MIHFamide C-termini, a NPF and a sulfakinin each ending in -RYamide rather than -RFamide, a precursor whose orcokinins include C-terminally amidated isoforms, and a collection of TRPs possessing -FXPXLamide rather than the stereotypical -FXGXLamide C-termini. The L. hesperus peptidome is by far the largest thus far published for any member of the Chelicerata. Taken collectively, these data serve as a reference for future neuropeptide discovery in the Araneae and provide a foundation for future studies of peptidergic control in L. hesperus and other spiders.

Topics: Amino Acid Sequence; Animals; Black Widow Spider; Computer Simulation; FMRFamide; Insect Hormones; Insect Proteins; Invertebrate Hormones; Molecular Sequence Data; Neuropeptides; Oligopeptides; Proteome; Transcriptome

The Aphidoidea is an insect superfamily comprising most of the known aphid species. While small in size, these animals are of considerable economic importance as many members of this taxon are serious agricultural pests, inflicting physical damage upon crop plants and serving as vectors in the transmission of viral plant diseases. In terms of identifying the paracrines/hormones used to modulate behavior, particularly peptides, members of the Aphidoidea have largely been ignored, as it is not tractable to isolate the large pools of tissue needed for standard biochemical investigations. Here, a bioinformatics approach to peptide discovery has been used to overcome this limitation of scale. Specifically, in silico searches of publicly accessible aphidoidean ESTs were conducted to identify transcripts encoding putative peptides precursors, with the mature peptides contained within them deduced using peptide processing software and homology to known arthropod sequences. In total, 39 ESTs encoding putative peptides precursors were identified from four aphid species: Acyrthosiphon pisum (14 ESTs), Aphis gossypii (four ESTs), Myzus persicae (20 ESTs) and Toxoptera citricida (one EST). These precursors included ones predicted to encode isoforms of B-type allatostatin, crustacean cardioactive peptide, FMRFamide-related peptide (both myosuppressin and short neuropeptide F subfamilies), insect kinin, orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide and tachykinin-related peptide. In total, 83 peptides were characterized from the identified precursors, most novel, including two B-type allatostatins possessing the variant -WX(7)Wamide motif, two N-terminally extended proctolin isoforms and an N-terminally truncated and substituted SIFamide. Collectively, these results expand greatly the number of known/predicted aphid peptide paracrines/hormones, and provide a strong foundation for future molecular and physiological investigations of peptidergic control in this insect group.

Topics: Amino Acid Sequence; Animals; Aphids; Computational Biology; Expressed Sequence Tags; FMRFamide; Insect Hormones; Molecular Sequence Data; Neuropeptides; Oligopeptides; Peptide Hormones; Sequence Homology, Amino Acid; Tachykinins

It is now well established that a dynamic balance of neurotransmitters and neuromodulators finely influence the output of neuronal networks and subsequent behaviors. In the present study, to further understand the modulatory processes that control locomotor behavior, we investigated the action of 11 neuropeptides, chosen among the various peptide subfamilies, on the lumbar neuronal network in the in vitro neonatal rat spinal cord preparation. Peptides were bath-applied alone, in combination with N-methyl-D,L-aspartate (NMA) or with the classical 'locomotor cocktail' of NMA and serotonin. Using these different experimental paradigms, we show that each peptide can neuromodulate the lumbar locomotor network and that peptides exhibit different neuromodulatory profiles and potencies even within the same family. Only vasopressin, oxytocin, bombesin and thyrotropin releasing hormone triggered tonic or non-organized rhythmic activities when bath-applied alone. All the neuropeptides modulated NMA induced activity and/ or ongoing sequences of fictive locomotion to varying degrees. These results suggest that neuropeptides play an important role in the control of the neural network for locomotion in the neonatal rat. Their various profiles of action may account in part for the great flexibility of motor behaviors.

Topics: Angiotensin II; Animals; Animals, Newborn; Bombesin; Bradykinin; Drug Synergism; Enkephalin, Methionine; FMRFamide; Ganglia, Spinal; Locomotion; Lumbosacral Region; Membrane Potentials; Motor Activity; N-Methylaspartate; Nerve Net; Neuropeptides; Neurotensin; Neurotransmitter Agents; Oligopeptides; Oxytocin; Rats; Rats, Wistar; Serotonin; Spinal Cord; Thyrotropin; Vasopressins

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Topics: Animals; Arrestins; beta-Arrestins; Cell Line; Cloning, Molecular; Cyclic AMP; Dose-Response Relationship, Drug; Drosophila; Drosophila Proteins; FMRFamide; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insect Hormones; Insect Proteins; Ligands; Luminescent Proteins; Microscopy, Confocal; Neuropeptides; Oligopeptides; Peptides; Protein Transport; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Receptors, Peptide; Receptors, Tachykinin; Transfection

Proctolin (Arg-Tyr-Leu-Pro-Thr-OH) and crayfish peptide "DF(2)" (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH(2)) enhance spontaneous contractions of isolated crayfish hindguts. Both peptides increase the frequency and amplitude of spontaneous, rapid contractions. Proctolin induces a slow contraction, which gives the appearance of an increase in overall tonus. DF(2) has no such effect. To determine whether the peptides affect both longitudinal and circular muscles, hindguts were cut into longitudinal strips and into rings, and contractions were recorded from each. The longitudinal strips generated only rapid contractions, and both peptides increased the frequency and amplitude of such contractions without significantly altering tonus. Rapid contractions were observed in only 1 of 14 preparations of rings. Proctolin induced slow contractions in the rings, and DF(2) had no such effect. The results indicate that neuropeptides have different effects on circular and longitudinal muscles of hindgut.

Topics: Animals; Astacoidea; FMRFamide; Intestines; Muscle Contraction; Muscle, Skeletal; Muscle, Smooth; Neuropeptides; Oligopeptides

Most crustacean muscle fibers receive double excitatory innervation by functionally different motor neurons termed slow and fast. By using specific omega-toxins we show that the terminals of the slow closer excitor (SCE) and the fast closer excitor (FCE) at a crab muscle are endowed with different sets of presynaptic Ca(2+) channel types. omega-Agatoxin, a blocker of vertebrate P/Q-type channels, reduced the amplitude of EPSCs by decreasing the mean quantal content of transmitter release in both neurons by 70-85%, depending on the concentration. We provide the first evidence that omega-conotoxin-sensitive channels also participate in transmission at crustacean neuromuscular terminals and are colocalized with omega-agatoxin-sensitive channels in an axon-type-specific distribution. omega-Conotoxin, a blocker of vertebrate N-type channels, inhibited release by 20-25% only at FCE, not at SCE endings. Low concentrations of Ni(2+), which block vertebrate R-type channels, inhibited release in endings of the SCE by up to 35%, but had little effects in FCE endings. We found that two neuropeptides, the FMRFamide-like DF(2) and proctolin, which occur in many crustaceans, potentiated evoked transmitter release differentially. Proctolin increased release at SCE and FCE endings, and DF(2) increased release only at FCE endings. Selective blocking of Ca(2+) channels by different omega-toxins in the presence of peptides revealed that the target of proctolin-mediated modulation is the omega-agatoxin-sensitive channel (P/Q-like), that of DF(2) the omega-conotoxin-sensitive channel (N-like). The differential effects of these two peptides allows fine tuning of transmitter release at two functionally different motor neurons innervating the same muscle.

Topics: Animals; Axons; Brachyura; Calcium Channel Blockers; Calcium Channels; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; FMRFamide; In Vitro Techniques; Muscles; Neuromuscular Junction; Neurons; Neuropeptides; Neurotransmitter Agents; Nickel; Oligopeptides; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Synaptic Transmission

After the extirpation of the brain reorganization of the peptidergic (FMRFamide, neuropeptide Y, proctolin) systems was studied in the newly forming cerebral ganglion of the annelid Eisenia fetida. During regeneration, all immunoreactive fibres appear on the 1st-2nd postoperative day. At the beginning of regeneration, immunoreactive neurons and fibres form a mixed structure in the wound tissue. On the 3rd postoperative day, FMRFamide positive and neuropeptide Y-immunoreactive, while on the 7th postoperative day proctolin-immunoreactive neurons appear in the loose wound tissue. From the 25th postoperative day a capsule gradually develops around it. The neurons of the preganglion move to the surface of the newly appearing preganglion. The number of these cells gradually increase, and by the 72th-80th postoperative days the localization and number of peptide-immunoreactive neurons is similar to that in the intact one. The neurons of all examined peptidergic systems may originate from the neuroblasts, situated on the inner and outer surface of the intact ganglia (e.g. suboesophageal and ventral cord ganglia). In addition FMRFamide and proctolin immunoreactive neurons may take their derive by mitotic proliferation from the pharyngeal neurons, too.

Topics: Animals; Brain; FMRFamide; Ganglia; Neuropeptide Y; Neuropeptides; Oligochaeta; Oligopeptides; Regeneration

We have previously shown differential expression of leucomyosuppressin (LMS) mRNA in apparent endocrine cells in the anterior region of midguts of the cockroach Diploptera punctata, using in situ hybridization. In contrast, other FMRFamide-related peptides, as revealed by immunohistochemistry, have been found most abundantly in the posterior region in both apparent endocrine cells and nerve tracts. Here, we partially purified extracts of anterior and posterior cockroach midguts, using HPLC coupled with radioimmunoassay, and found, among multiple FMRFamide-like immunoreactive fractions, one fraction co-eluting with LMS in both regions. The presence of a co-eluting fraction in the posterior region, in the absence of LMS mRNA positive endocrine cells suggests that LMS might therefore be present in nerve tracts running along the length of the midgut. Using a circular muscle contraction assay from different portions of midgut, we determined the effects of LMS, proctolin and a variety of other midgut peptides on contractions of the midgut of Diploptera. Proctolin caused a sustained tonic contraction in the anterior midgut, the amplitude of which was dose-dependent. In contrast, LMS, and its relative SchistoFLRFamide, reduced the amplitude of these contractions. LMS and SchistoFLRFamide also inhibited spontaneous phasic contractions, which were elicited by proctolin application in only a few preparations. Other postulated midgut peptides did not induce or inhibit contractions, nor augment the proctolin-induced contractions. The C-terminal truncated sequences of LMS, HVFLRFamide and VFLRFamide, were sufficient to reduce the amplitude of the proctolin-induced contractions. This work illustrates a possible physiological role for LMS in Diploptera midguts, in the passage of food along the alimentary canal.

Topics: Animals; Cockroaches; FMRFamide; Muscle Contraction; Nerve Tissue Proteins; Neuropeptides; Oligopeptides; Peptide Fragments; Peptides

The role of neurotransmitters in the modulation of the sodium-dependent high affinity choline uptake system (HAChUS) of the horseshoe crab, Limulus polyphemus has been investigated utilizing a tissue slice preparation. Choline uptake was significantly decreased by carbachol but unaffected by atropine and d-tubocurarine. The muscarinic agonist oxotremorine decreased choline uptake by 30.4% while the muscarinic antagonist, pirenzepine, increased uptake by 29.6%. Applied in combination, pirenzepine and oxotremorine abolished their individual effects resulting in control values for choline uptake. The non-cholinergic transmitters octopamine and serotonin significantly enhanced choline uptake. The neuropeptide proctolin elicited a 20% increase in choline transport whereas Phe-Met-Arg-Phe (FMRF) amide was without effect. This study demonstrates that neurotransmitters and neuropeptides modulate the HAChUS, possibly through specific receptor-mediated second messenger systems.

Topics: Animals; Atropine; Brain; Carbachol; Choline; FMRFamide; Horseshoe Crabs; Neuropeptides; Neurotransmitter Agents; Octopamine; Oligopeptides; Oxotremorine; Pirenzepine; Potassium; Serotonin; Tubocurarine

1. The morphology and innervation of the midgut (intestine) in the horseshoe crab, Limulus polyphemus was investigated. The organization of this tissue was examined with routine histology. Radioimmunoassay, immunohistochemistry and high performance liquid chromatography were employed to detect, localize and identify peptidergic innervation of the midgut. The actions of synthetic and native proctolin-like and FMRFamide-like peptides were compared on the isolated midgut preparation. 2. Levels of proctolin and FMRFamide were determined in extracts of Limulus midgut tissue using radioimmunoassay. High levels of proctolin-like immunoreactivity (69.5 +/- 11.3 ng/g) were detected, while levels of FMRFamide-like immunoreactivity (0.8 +/- 0.2 ng/g) were less. Proctolin levels were equally distributed, while the levels of FMRFamide-like immunoreactivity exhibited an anterior bias. 3. Proctolin- and FMRFamide-like immunoreactivities in the Limulus midgut were localized with immunohistochemistry. Proctolin- and FMRFamide-immunoreactive elements were detected in intestinal nerve branches and individual fibers running along the surface of the midgut in whole-mount preparations. In sectioned tissue, staining for these peptides was observed throughout the midgut, typically associated with muscle bands and fibers. Only a few immunoreactive cell bodies were observed. 4. Proctolin, and several FMRFamide-like peptides produced distinct and opposing actions on the isolated Limulus midgut preparation. Proctolin elicited contracture and rhythmic contractions of this tissue, while FMRFamide and N-terminally extended analogs of FLRFamide relaxed gut tension. FMRFamide-like peptides partially reversed the excitatory actions of proctolin. 5. Proctolin- and FMRFamide-like peptides in Limulus midgut extracts were partially characterized with high performance liquid chromatography. One peak of proctolin-like activity was detected on a linear gradient of 18 to 31.5% acetonitrile. The native proctolin-like peptide produced excitatory actions on the isolated midgut preparation which were indistinguishable from those produced by synthetic proctolin. Several peaks of FMRFamide-like bioactivity (Busycon radula protractor muscle assay) were detected with a linear gradient of 5 to 30% acetonitrile. Fractions from two distinct peaks produced FMRFamide-like inhibitory effects on the isolated Limulus midgut preparation. These findings suggest a role for proctolin-like and FMRFamide-like peptides

Topics: Animals; Chromatography, High Pressure Liquid; FMRFamide; Horseshoe Crabs; Immunohistochemistry; Intestines; Mollusca; Muscles; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Peptides; Radioimmunoassay

The pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion receive modulatory inputs from more anteriorly located ganglia via the stomatogastric nerve. In this study we employed biocytin backfilling and immunostaining, as well as electron microscopy, to determine the origin of these inputs in the crab, Cancer borealis. Fiber counts from electron micrographs of sections through the stomatogastric nerve showed that this nerve contains 55-60 medium to large diameter fibers (1-13 microns). These fibers were individually wrapped by several layers of membrane, presumably glial in origin. There was also a single cluster of jointly wrapped, small diameter (< 1 micron) fibers that may originate from peripheral sensory somata. Biocytin backfills revealed that approximately two thirds of the individually wrapped fibers in this nerve originate from somata in the other three ganglia of the stomatogastric nervous system, including the paired commissural ganglia and the single oesophageal ganglion. There were approximately 20 biocytin-labeled somata in each commissural ganglion and 3 somata in the oesophageal ganglion. An additional ten somata were localized to the stomatogastric ganglion itself. This accounts for nearly all of the medium to large diameter fibers in the stomatogastric nerve. We used double-labeling with backfills and immunocytochemistry to determine that there are two proctolin-immunoreactive neurons and four FMRFamide-like immunoreactive neurons among the biocytin-labeled neurons in each commissural ganglion. Both peptides modulate neural network activity in the stomatogastric ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brachyura; Fluorescent Antibody Technique; FMRFamide; Ganglia; Histocytochemistry; Lysine; Microscopy, Electron; Neuropeptides; Neurotransmitter Agents; Oligopeptides

1. The innervation of the hindgut, rectal valve, rectum and rectal papillae of the adult blowfly, Calliphora erythrocephala, was studied by means of light and electron microscopic immunocytochemistry, using antibodies against the neuropeptides proctolin and FMRFamide. 2. Branches from the abdominal nerves reaching the posterior portion of the gut were found to contain mostly neurosecretory type axons and to innervate the muscle coat of all hindgut structures studied. 3. Some of the axons found in these nerve branches innervating the gut display proctolin- others FMRFamide-like immunoreactivity. Both types of peptidergic axons were found to have abundant terminals in the muscle coat of the hindgut, rectum and rectal valve and in the medulla of the rectal papillae. 4. It is clear that two separate peptidergic systems derived from the abdominal ganglion are supplying the hindgut structures, and, possibly, they use proctolin- and FMRFamide-like peptides respectively as their transmitters or modulators.

Topics: Animals; Digestive System; Diptera; FMRFamide; Immunohistochemistry; Microscopy, Electron; Neurons, Efferent; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Rectum

Topics: Amino Acid Sequence; Animals; Digestive System; Drug Interactions; Female; FMRFamide; Grasshoppers; In Vitro Techniques; Male; Molecular Sequence Data; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Peptides; Serotonin

1. The effects of synthetic biologically active peptides, including Met-enkephalin, substance P, oxytocin, Arg-vasopressin, proctolin and FMRFamide, on the following four buccal neurones were examined: d-LBAN (dorsal-left buccal anterior neurone), d-LBMN (dorsal-left buccal medial neurone), d-LBCN (dorsal-left buccal central neurone) and d-LBPN (dorsal-left buccal posterior neurone). These peptides were examined at 10(-4) M. 2. Oxytocin excited d-LBAN and slightly excited d-LBCN, while this inhibited d-LBMN. Arg-vasopressin excited slightly d-LBAN and d-LBCN, but this had some times no effect. FMRFamide inhibited d-LBAN, and slightly inhibited d-LBCN. 3. No direct synaptic connection from the two ventral cerebral giant neurones, v-LCDN and v-RCDN, to the four buccal giant neurones was found, though the two cerebral neurones innervate the cerebro-buccal connectives.

Topics: Animals; Arginine Vasopressin; Cheek; FMRFamide; Ganglia; Hormones; In Vitro Techniques; Membrane Potentials; Neurons; Neuropeptides; Oligopeptides; Oxytocin; Peptides; Snails; Synaptic Membranes

Effects of neuropeptides proposed as neurotransmitters of mammals and invertebrates and those of natural toxins, etc., on the identifiable giant neurones of an African giant snail (Achatina fulica Férussac) were examined. Oxytocin had a tendency to show the excitatory effects; this substance was excitatory on five neurones and inhibitory on one neurone. A substance related to oxytocin, vasotocin, also showed excitatory effects on the three neurones. On the other hand, FMR Famide was inhibitory on six neurones. Proctolin had excitatory effects on only one neurone.

Topics: Amino Acid Sequence; Animals; FMRFamide; Molecular Sequence Data; Neurons; Neuropeptides; Oligopeptides; Oxytocin; Snails; Vasotocin

Thirteen synthetic biologically-active peptides, which were classified into the peptides proposed as neurotransmitters in mammals and invertebrates and neural venom peptides, were investigated for their effects on the following six identifiable giant neurons of an African giant snail (Achatina fulica Férussac): RAPN (right anterior pallial neuron), INN (intestinal nerve neuron), RPeNLN (right pedal nerve large neuron), LPeNLN (left pedal nerve large neuron), d-LPeLN (dorsal-left pedal large neuron) and d-LPeCN (dorsal-left pedal constantly firing neuron). Oxytocin and proctolin at 10(-4)M excited the RAPN membrane potential, whereas FMRFamide at the same concentration inhibited the same neuron. FMRFamide at 10(-4)M markedly inhibited the d-LPeLN membrane potential, sometimes produced inhibition of RPeNLN and LPeNLN, showed varied effects (excitatory or inhibitory) on INN, and had no effect on d-LPeCN. The other peptides examined had almost no effect on any of the neurons tested.

Topics: Animals; FMRFamide; Ganglia; Membrane Potentials; Neurons; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Oxytocin; Peptides; Snails

The neuropeptides, proctolin and FMRFamide, increase the frequency of, and modify the motor pattern produced by, the stomatogastric ganglion (STG) of the crab, Cancer irroratus. Both proctolin-like and FMRFamide-like immunoreactivities are present in fibers in the stomatogastric nerve which terminate in the neuropile of the STG. The neural output of the STG thus appears to be modulated by at least two different groups of peptidergic input fibers.

Topics: Animals; Brachyura; FMRFamide; Ganglia; In Vitro Techniques; Motor Neurons; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Stimulation, Chemical