fmrfamide and phenylalanyl-leucyl-arginyl-phenylalaninamide

From a single LC-MS/MS analysis, a new C-terminally extended RFamide neuropeptide was characterized in Sepia officinalis. The experimental strategy was based on the specific neutral loss associated with RFamide breakdown. Mass losses of 17 Da (C-terminally amide) and 320 Da (RFamide) have been observed for three known peaks of m/z 581.7 (FLRFamide), 599.8 (FMRFamide), 1096.3 (ALSGDAFLRFamide) and one unknown of m/z 752.8. The primary sequence of the peptide of m/z 752.8 was GNLFRFamide. MS/MS analyses revealed that this novel neuropeptide, called sepFRF1, is largely distributed in the central nervous system of cuttlefish of both sexes. Probably transported in the visceral nerve from the subesophageal mass (the peptide was not detected in the hemolymph), this neuropeptide targeted the rectum in agreement with its peripheral distribution. From concentrations as low as 10(-9)M, sepFRF1 increased the frequency, tonus and amplitude of rectal contractions. SepFRF1 is the first RFamide peptide identified in Sepia officinalis that is not derived from the FaRPs precursor. SepFRF1 could belong to a RFamide subfamily identified in gastropods and may be involved in feeding behavior.

Topics: Amino Acid Sequence; Animal Structures; Animals; Central Nervous System; Female; FMRFamide; Male; Muscle Tonus; Muscle, Smooth; Nerve Endings; Neuropeptides; Oligopeptides; Oviducts; Rectum; Sepia; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

FMRFamide and the related tetrapeptide FLRFamide are highly excitatory in molluscan non-cardiac smooth muscle. They are also exceptionally excitatory in the atrium and internally perfused ventricle of Busycon canaliculatum. These two peptides, usually thought of as classic molluscan cardio-acceleratory agents are in fact simply two members of a large and ever growing superfamily, the RFamide family, whose phylogenetic distribution has been so elegantly mapped by Walker. Members of this family, often with extended peptide chains (e.g. penta, hepta and decapeptides), stretch in their known distribution from the cnidaria to the chordates. The effects of some of the members of this superfamily (FMRFamide. FLRFamide, YMRFamide, TNRNFLRFamide, SDPFLRFamide, LMS) were examined. The neuropeptides were found to be very potent at very low concentrations (10(-9) M) in the ventricle of both Buccinium and Busycon. Other neuropeptides (HFMRdFamide, SCPb, NLERFamide and pEGRFamide) were found to be without any effect. The Ca2+ dependency of these neuropeptides was also tested. The peptides appear to induce contraction of the ventricles by release of Ca2+ from internal pools. The neuropeptides appear to stimulate contraction in these cardiac muscles through a completely different pathway to Serotonin (the main excitatory neurotransmitter for the cardiac muscle). When the peptides were applied together with Serotonin an additive effect was observed clearly indicating the release of Ca2+ through different pathways. The nature of the RFamide receptor was also tested. It appears that the RFamide neuropeptides mobilize the 2nd messenger IP3 (Inositol trisphosphate), since the IP3 blocker Neomycin Sulphate inhibited the response of the neuropeptides.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Electrophysiology; FMRFamide; Inositol Phosphates; Mollusca; Myocardium; Neomycin; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Peptides; Protein Synthesis Inhibitors; Seawater; Species Specificity; Time Factors

In this study the relative potencies of four established molluscan cardioexcitatory agents were examined on Buccinum heart. The potencies were, in decending order: phenylalanine-leucine-arginine-phenylalanine-NH2 (FLRFamide) > phenylalanine-methionine-arginine-phenylalanine-NH2 (FMRFamide; 80% of maximum) > 5-hydroxytryptamine (5HT; 60% of maximum) > guanosine triphosphate (GTP; 15% of maximum). FMRFamide and FLRFamide had similar dose-response curve patterns with thresholds at 10(-9) mol l(-1) but FLRFamide was more potent than FMRFamide. The superfused atrium was much less sensitive to all agonists than the internally perfused ventricle. FLRFamide and FMRFamide induced small depolarizations (1-2 mV) which triggered a burst of action potentials of about 5 mV which on reaching 4 mV triggered a burst of fast twitch contractions. Lithium, at high concentrations inhibited FMRFamide and 5-HT responses of internally perfused ventricles. Neomycin also inhibited peptide responses, but was without effect on 5-HT responses. Heparin, however, for technical reasons was without effect on ventricular responses to all three agonists. FMRFamide and FLRFamide appear to share a common receptor, the potency difference being due to the substitution of leucine for methionine in FLRFamide. The RF N-terminal sequence appears crucial for receptor activation. The Phospholipase C inhibitor neomycin equally inhibits responses to the two peptides while 5-HT responses are unaffected. This implicates a peptide/receptor interaction which activated inositol 1,4,5-trisphosphate (IP3) as a second messenger.

Topics: Animals; Anticoagulants; Antimanic Agents; Bivalvia; Dose-Response Relationship, Drug; FMRFamide; Guanosine Triphosphate; Heparin; Inositol 1,4,5-Trisphosphate; Lithium Chloride; Membrane Potentials; Myocardial Contraction; Myocardium; Neomycin; Oligopeptides; Protein Synthesis Inhibitors; Second Messenger Systems; Serotonin; Type C Phospholipases

Pharmacological study of Phe-Met-Leu-Phe-amide (FMRFa) receptors is hindered by the lack of selective ligands. The classification of these selective ligands is further hampered by the limited availability of functional assays. In this study, we evaluated several synthetic FMRFa analogs for agonist and antagonist activity by measuring their abilities to produce [35-S]-GTP-gamma-S stimulation or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding in squid optic lobes. Analogs included acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa), desamino-Tyr-Phe-Leu-Arg-amide (daYFLRa), desamino Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa), desamino Tyr-Phe-norLeu-Arg-[TIC]-amide (daYFnLR[TIC]a), desamino Tyr-Trp-norLeu-Arg-amide (daYWnLRa), (D)-Tyr-Phe-norLeu-Arg-Phe-amide (D)-YFnLRFa), Phe-Leu-Arg-Phe-amide (FLRFa), and the D-amino acid analogs of FMRFa (D-FMRFa, F-(D)-MRFa and FM-(D)-RFa). For agonist studies, full dose-response curves were generated and analyzed for potency and efficacy (maximal percent effect). FMRFamide as well as analogs ac-FnLRFa, daYFnLRFa, daYFnLR[TIC]a, D-YFnLRFa, FLRFa, and (D)-FMRFa stimulated [35S]-GTP-gamma-S binding. Analogs daYWnLRa, daYFLRa, F-(D)-MRFa, and FM-(D)-RFa failed to stimulate either [35S]-GTP-gamma-S binding or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding. The rank order of potency was daYFnLRFa > or = daYFnLRF[TIC]a > acFnLRFa > (D)YFnLRFa > FLRFa > or = FMRFa >> (D)-FMRFa. The order of efficacy was daYFnLRFa = acFnLRFa = (D)-YFnLRFa > FLRFa = FMRFa > or = (D)-FMRFa > or = daYFnLRF[TIC]a. Peptide analog daYFnLR[TIC]a was less efficacious (59% maximal stimulation) than analogs daYFnLRFa, acFnLRFa, and (D)-YFnLRFa (113-146% maximal stimulation). A maximal concentration of daYFnLR[TIC]a (10 microM) reduced daYFnLRFa, acFnLRFa, and (D)-YFnLRFa induced [35S]-GTP-gamma-S stimulation, indicating that daYFnLR[TIC]a is a partial agonist at the receptor stimulated by the FMRFamide analogs. Analysis of the structural requirements needed for promoting [35S]-GTP-gamma-S binding show that elongation (i.e., daYFnLRFa, D-YFnLRFa) or modification of Phe1 (ac-FnLRFa) leads to increased efficacy and potency. Moreover, elimination of the C-terminal Phe (daYWnLRa, daYFLRa,) leads to a loss of biological activity. However, substitution with L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid, a rigid analog of the C-terminal Phe (daYFnLR[TIC]a), leads to decreased efficacy but not loss of potency. The data suggest that immobilization or modification

Topics: Animals; Decapodiformes; Drug Evaluation, Preclinical; FMRFamide; Guanosine 5'-O-(3-Thiotriphosphate); Membranes; Oligopeptides; Optic Lobe, Nonmammalian; Radioligand Assay; Receptors, Invertebrate Peptide; Subcellular Fractions

1. Single-channel recordings were made from outside-out membrane patches of Xenopus oocytes injected with the cDNA clone FaNaCh, which encodes a peptide-gated Na+ channel from Helix aspersa. 2. The natural peptides FMRFamide and FLRFamide only activated unitary currents in oocytes injected with FaNaCh; the EC50 values were 1.8 and 11.7 microM, respectively. 3. The slope conductance of the channel was 9.2 pS for both peptides. 4. With FMRFamide, the open probability (Po) of the channel was 0.06 at 0.3 microM and 0.76 at 30 microM, whereas for FLRFamide the open probability increased from 0.04 at 1.8 microM to 0.49 at 50 microM. The Hill coefficient was greater than 1 for both peptides. 5. High concentrations of each peptide evoked very fast flickering between open and closed states which led to decreased unitary current amplitude. 6. At low doses, brief single openings and bursts of longer openings occurred. With higher doses, the occurrence of the brief openings declined and the number of longer openings increased; the duration of the longer openings was shorter with FLRFamide than with FMRFamide. 7. For each peptide, frequency distribution histograms of open events were best fitted by the sum of two exponential components, suggesting the existence of two open states of the channel. Closed events were fitted by the sum of three components, suggesting the existence of three closed states. 8. The data were analysed according to a five-state model in which the brief openings correspond to a single liganded open form of the channel and the longer openings to a doubly liganded open form. According to this interpretation, the greater whole-cell response observed with FMRFamide than with FLRFamide results mostly from a slower closing rate constant for the longer (doubly liganded) channel openings.

Topics: Animals; Biotransformation; Electric Stimulation; Electrophysiology; FMRFamide; Helix, Snails; Ion Channel Gating; Membrane Potentials; Oligopeptides; Oocytes; Patch-Clamp Techniques; Peptides; RNA, Messenger; Sodium Channels; Xenopus

The endogenous neuropeptides FMRFamide and FLRFamide (tetrapeptides) reversibly reduced a voltage-activated calcium current in the C1 neuron of Helix aspersa by an average of 20%. Two structurally related heptapeptides, pQDPFLRFamide and pQDPFLRIamide, both derived from another precursor protein in this species, did not reduce the current at all.

Topics: Animals; Calcium Channels; Enkephalin, Methionine; Evoked Potentials; FMRFamide; Helix, Snails; Neurons; Neuropeptides; Oligopeptides; Pyrrolidonecarboxylic Acid

The actions of known platyhelminth FaRPs on the contractility of whole-worm preparations of the monogenean, Diclidophora merlangi have been examined in vitro for the first time. All of the peptides tested had excitatory effects on the motor activity of the worm. The order of potency for the peptides tested was: YIRFamide > GYIRFamide = RYIRFamide > GNFFRFamide = FLRFamide. However, although YIRFamide was more potent than GYIRFamide, the latter was the most efficacious on each of the motility parameters (tension, contraction amplitude and contraction frequency) examined at concentrations > or = 0.1 microM. Serotonin, which stimulates contractility in the worm was used as a positive control. The excitatory activity of turbellarian and cestode neuropeptides on a monogenean indicates at least some structural similarities in the neuropeptide receptors of these classes of flatworm.

Topics: Animals; Fishes; FMRFamide; Gills; Helminth Proteins; Movement; Neuropeptides; Oligopeptides; Platyhelminths; Serotonin; Structure-Activity Relationship

Three FMRFamide-related peptides (FaRPs) were purified and characterized from the kidney of the snail, Helisoma trivolvis, by HPLC and detected using two radioimmunoassays (RIA) for FaRPs. Automated sequencing and mass spectrometry of the isolated peptides suggest the following sequences: Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FMRFamide), and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide). The FaRPs, predominantly the heptapeptides, were also detected by HPLC and RIA in other osmoregulatory tissues such as the skin, mantle, and the hemolymph. The level of FaRPs, detected by radioimmunoassay, appears to be lower in snails kept under hyposmotic stress than in snails kept under isosmotic stress. The FaRPs appear to be involved in osmoregulation in H. trivolvis.

Topics: Amino Acid Sequence; Animals; FMRFamide; Hemolymph; Invertebrate Hormones; Kidney; Molecular Sequence Data; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Organ Specificity; Skin; Snails; Water-Electrolyte Balance

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.

Topics: Age Factors; Amino Acid Sequence; Animals; Central Nervous System; FMRFamide; Ganglia, Invertebrate; Helix, Snails; Molecular Sequence Data; Neurons; Neuropeptides; Oligopeptides; Pyrrolidonecarboxylic Acid; RNA, Messenger

Peptidergic regulation of the contraction of radula muscles and the heart beat in a prosobranch mollusc, Rapana thomasiana was investigated. In the radula muscles, FMRFamide-related peptides were suggested to act on presynaptic sites to enhance their contraction elicited by nerve stimulation, possibly by increasing the release of a main transmitter. A catch-relaxing peptide (CARP) was demonstrated to depress the contraction by acting on postsynaptic sites. In the heart, FMRFamide enhanced the amplitude and frequency of the beat, whereas CARP depressed it. Immunohistochemical studies of the buccal ganglia have revealed that FMRFamide- and CARP-like immunoreactive neurons are distributed in the same region, indicating a possible coexistence of the both peptides. In the visceral ganglia, on the contrary, coexistence of FMRFamide and CARP was not observed. FMRFamide- and CARP-like immunoreactivities were also detected in the nerve fibers in the radula muscles and the atria. The present results show that FMRFamide- and CARP-like peptides are involved in the regulation of the contraction of radula muscles and the heart beat.

Topics: Amino Acid Sequence; Animals; FMRFamide; Ganglia; Molecular Sequence Data; Mollusca; Muscle Contraction; Myocardial Contraction; Neurons; Neuropeptides; Oligopeptides

The stellate ganglion of the squid Loligo pealli contains the neuropeptides Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FLRFamide) and at least one N-terminally extended FMRFamide-related peptide that is yet to be fully characterized. Both local application and arterial perfusion of FLRFamide potentiate transmission at the giant synapse. The N-terminally related peptide Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) produced a similar effect. The threshold for both the tetra- and the hepta-peptides was less than 10 microM. Potentiation could be detected as an increase in rate of rise of the EPSPs, as an increase in amplitude of the EPSP in the absence of spikes, or under voltage clamp as an increase in the EPSC. The effect was most pronounced when the synapse was fatigued by high frequency stimulation. Another molluscan peptide, eledoisin and also leucine enkephalin were without effect. In the absence of any detectable effects of FLRFamide on the resting membrane potential of either pre- or postsynaptic terminals or on the presynaptic spike, it is suggested that the peptide influences transmitter mobilization. However, the peptide could also exert small changes in preterminal calcium currents, which so far we have been unable to detect.

Topics: Animals; Arteries; FMRFamide; Microinjections; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Perfusion; Stellate Ganglion; Synapses; Synaptic Transmission