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Peptide F1, an N-terminally extended analog of FMRFamide, enhances contractile activity in multiple target tissues in lobster.

The physiological actions of lobster peptide F1 (TNRNFLRFamide) have been examined on three different lobster nerve-muscle preparations (exoskeletal, cardiac and visceral). The peptide, which is found at high concentrations in a lobster neurosecretory gland, causes a long-lasting enhancement of contractility in each target tissue. On exoskeletal nerve-muscle preparations, peptide F1 has the following actions: (1) it potentiates transmitter release from nerve terminals innervating exoskeletal muscle, leading to an increase in both spontaneous and nerve-evoked release of transmitter; (2) it acts directly on the muscle, in the absence of nerve activity, to induce tonic contractions; and (3) it shows a potent desensitization that does not reverse with prolonged washing of the tissue. On each of the types of muscle examined, peptide F1 is active at nanomolar concentrations and is 3-4 orders of magnitude more potent than FMRFamide. These findings suggest that peptide F1 is a neurohormone with widespread myogenic actions throughout lobster peripheral tissues. The molecular mechanism(s) by which the peptide acts are not yet known, but do not appear to involve cyclic AMP or cyclic GMP.

Amino Acid Sequence↗

Mass spectrometric survey of peptides in cephalopods with an emphasis on the FMRFamide-related peptides.

A matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) survey of the major peptides in the stellar, fin and pallial nerves and the posterior chromatophore lobe of the cephalopods Sepia officinalis, Loligo opalescens and Dosidicus gigas has been performed. Although a large number of putative peptides are distinct among the three species, several molecular masses are conserved. In addition to peptides, characterization of the lipid content of the nerves is reported, and these lipid peaks account for many of the lower molecular masses observed. One conserved set of peaks corresponds to the FMRFamide-related peptides (FRPs). The Loligo opalescens FMRFa gene has been sequenced. It encodes a 331 amino acid residue prohormone that is processed into 14 FRPs, which are both predicted by the nucleotide sequence and confirmed by MALDI MS. The FRPs predicted by this gene (FMRFa, FLRFa/FIRFa and ALSGDAFLRFa) are observed in all three species, indicating that members of this peptide family are highly conserved across cephalopods.

Amino Acid Sequence↗

A statistical view of FMRFamide neuropeptide diversity.

FMRFamide-like peptide (FLP) amino acid sequences have been collected and statistically analyzed. FLP amino acid composition as a function of position in the peptide is graphically presented for several major phyla. Results of total amino acid composition and frequencies of pairs of FLP amino acids have been computed and compared with corresponding values from the entire GenBank protein sequence database. The data for pairwise distributions of amino acids should help in future structure-function studies of FLPs. To aid in future peptide discovery, a computer program and search protocol was developed to identify FLPs from the GenBank protein database without the use of keywords.

Amino Acid Sequence↗

Excitatory actions of FMRFamide-related peptides (FaRPs) on the neurogenic Limulus heart.

The actions of FMRFamide-related peptides (FaRPs) on the neurogenic heart of the horseshoe crab, Limulus polyphemus, were investigated. Excitatory chronotropic effects were produced by application of TNRNFLRFamide, SDRNFLRFamide, GYNRS-FLRFamide, or pQDPFLRFamide to the intact heart preparation. Effects were dose-dependent with a threshold of 10(-9) M or less. TNRNFLRFamide and SDRNFLRFamide increased the burst rate of the isolated Limulus cardiac ganglion. Synthetic FaRPs produced inotropic excitation of the heartbeat as well. GYNRSFLRFamide, TNRNFLRFamide, SDRNFLRFamide, and pQDPFLRFamide increased heart contraction strength at a threshold dose of approximately 10(-8) M. TNRNFLRFamide and SDRNFLRFamide enhanced electrically evoked contractions of the Limulus myocardium, elicited contracture in some preparations, and increased the excitability of cardiac muscle fibers. The presence of cardioactive FaRPs in the Limulus central nervous system was suggested by reverse phase HPLC of acidified methanol extracts of Limulus nervous tissue. Four peaks of FaRP-like bioactivity were detected with the Busycon radula protractor muscle bioassay. These peaks also contained FaRP-like immunoreactivity. Two of these partially purified peaks produced excitatory chronotropic effects on the intact Limulus heart preparation similar to those produced by synthetic FaRPs.

Amino Acid Sequence↗

Osmoregulation and FMRFamide-related peptides in the salt marsh snail Melampus bidentatus (Say) (Mollusca: Pulmonata).

The pulmonate snail Melampus bidentatus occupies the high intertidal zone of salt marshes in a nearly terrestrial environment. The hemolymph osmolarity of the snails collected in the field paralleled that of the adjacent water and was affected by the tides and precipitation. The snails initially gained or lost weight when submerged in hypo- or hyperosmotic media, respectively, but returned to their original weight after 24 h. The content of their immunoreactive (IR)-FMRFamide-Related Peptides (FaRPs) was measured in various tissues by radioimmunoassay, and IR-FaRPs were found in every tissue analyzed. The subesophageal part of the central nervous system (CNS) contained more IR-FaRPs than the supraesophageal part, and the kidney and the tissues of the reproductive tract contained more than other peripheral tissues. The levels of IR-FaRPs in the CNS, kidney, and hemolymph were higher in snails that were immersed in higher concentrations of seawater. Many IR neurons are present in all ganglia of the CNS except the pleural ganglia, and IR neurites are extensively distributed within the CNS and its connective tissue sheath. The visceral nerve from the visceral ganglion is immunoreactive and could be seen to innervate the kidney, which contains IR-varicosities. An osmoregulatory role for the FaRPs is suggested.

Animals↗

Development of embryonic and larval cells containing serotonin, catecholamines, and FMRFamide-related peptides in the gastropod mollusc Phestilla sibogae.

The present immunocytochemical study provides one of the first detailed descriptions of the development of cells containing a variety of neurotransmitters during much of the larval life of a nudibranch gastropod. Throughout much of early development, serotonergic cells were located only in the apical organ; as larvae approached metamorphosis, serotonergic cells were also detected in the cerebropleural and pedal ganglia. Cells exhibiting tyrosine hydroxylase immunoreactivity (indicative of catecholamine synthesis) were first located near the mouth but by late embryonic stages were also located in the apical organ and near the velum and eyes. By late larval stages, numerous catecholaminergic cells were found in the foot, with concentrations in the propodium. Finally, the first cells exhibiting FMRFamide immunoreactivity were detected posterior to the neuropil of the cerebropleural ganglia in the early embryo. Fibers that presumably originated from these cells subsequently invaded the cerebral and pedal ganglia and the apical organ. By early larval stages, a second pair of peptidergic neurons was located near the first pair, and additional peptidergic neurons were located in the apical organ and peripheral positions in the foot and medial and dorsal to the eyes. In addition to providing a unique phyletic perspective to our understanding of gastropod neural development, the present study also sets the stage for future studies into changes in the nervous system as this gastropod undergoes metamorphosis.

Animals↗

Distribution and localization of immunoreactive FMRFamide-like peptides in the lancelet.

Immunofluorescence was used to study the distribution of FMRFamide (Phe-Met-Arg-Phe-NH2) in premetamorphic larvae and adults of the lancelet, Branchiostoma lanceolatum. In the larvae, FMR-Famide-containing presumably neuronal perikarya and fibers were limited to the anterior third of the dorsal nerve cord. Throughout this region, most of the immunoreactive perikarya and fibers were located ventrolaterally and ventrally within the nerve cord; in addition, in the caudal part of the cerebral vesicle, some of the immunofluorescent cells projected cytoplasmic extensions across the slot-like neural canal. In adult lancelets, immunofluorescence was detected in cells of the Hatschek's pit (a probable homologue of the anterior hypophysis of vertebrates); however, no immunofluorescence was detected in the larval preoral pit, which is the ontogenetic precursor of Hatschek's pit. Moreover, the FMR-Famide-containing elements do not show immunoreactivity to other peptides of the FaRPs family such as pancreatic polypeptide (PP). The results suggest that FMRF-amide may be involved in neuroendocrine functions of lancelets.

Animals↗

Establishment of the FMRFamide-immunoreactive olfacto-retinalis pathway during ontogeny of the rainbow trout (Oncorhynchus mykiss).

Migration of neurons is one of the mechanisms establishing normal central nervous system connectivity during ontogeny. Proper timing of axonal sprouting is relevant in the same context. In the present study, we used the immunoreactivity of the tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) to visualize the olfacto-retinalis projection during trout ontogeny. It starts to innervate the retina two to four weeks after hatching, in contrast to reports on salmon where it only appears after the fish are imprinted on their natal stream.

Aging↗

Effects of naloxone on c-jun/AP-1 in met-enkephalin-and FMRFamide-immunreactive neurons of a gastropod snail.

1. Opioid- and FMRFamide (FMRFa)-ergic systems are believed to play antagonistic behavioral roles in both higher and lower animals. In our previous experiments on a snail, behavioral choice has been demonstrated to be dependent on a balance between FMRFa and enkephalins [7]. Here, we examined if the disturbance of the balance causes changes in the activity of both systems. Opiate receptor blocker naloxone was applied and its effect on c-jun expression of met-enkephalin (MEnk)- and FMRFa-ergic neurons was examined immunocytochemically in terrestrial gastropod snail Cepaea nemoralis. 2. In control, untreated snails, central neurons with c-jun/AP-1-like-immunoreactivity were found to occur. These included MEnk-, FMRFa- and 5HT-immunoreactive (-ir) neurons, as was revealed by double-labelling. 3. After treatment with naloxone for 4 h, the following changes were observed: (i) increase in the number of MEnk-ir neurons; increase in the number of neurons showing c-jun/AP-1 and MEnk double-labeling; (ii) disappearance of c-jun/AP-1-immunoreactivity from some FMRFa-ir neurons. 4. It is suggested that immediate early genes are involved in the mechanisms responsible for the reciprocal regulation of the opioid and antiopioid neuropeptide systems.

Animals↗

Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain.

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.

Animals↗

Selective modulation of spike duration by serotonin and the neuropeptides, FMRFamide, SCPB, buccalin and myomodulin in different classes of mechanoafferent neurons in the cerebral ganglion of Aplysia.

An examination of the cellular properties and synaptic outputs of mechanoafferent neurons found on the ventrocaudal surface of the cerebral ganglion of Aplysia indicated that the cerebral mechanoafferent (CM) neurons are a heterogeneous population of cells. Based on changes in action potential duration in response to bath applications of 5-HT in the presence of TEA, CM neurons could be divided into 2 broad classes: mechanoafferents whose spikes broaden in response to 5-HT (CM-SB neurons) and mechanoafferents whose spikes narrow in response to 5-HT (CM-SN neurons). Morphological and electrophysiological studies of the CM-SN neurons indicated that they were comprised of previously identified interganglionic cerebral-buccal mechanoafferent (ICBM) neurons and a novel set of sensory neurons that send an axon into the LLAB cerebral nerve and have perioral zone receptive fields that are similar to those of ICBM neurons. Changes in spike width due to 5-HT were correlated with changes in synaptic output as indicated by the magnitudes of EPSPs evoked in postsynaptic neurons. Electrical stimulation of cerebral nerves and connectives also produced spike narrowing or broadening, and the sign of the effect was a function of the parameters of stimulation. Both heterosynaptic facilitation and heterosynaptic depression of EPSPs evoked in follower cells could be demonstrated. A variety of putative neuromodulators other than 5-HT were also found to affect the duration of action potentials in both classes of CM neurons. FMRFamide had effects opposite to that of 5-HT. SCPB and a recently characterized Aplysia neuropeptide, buccalin, broadened the spikes of both CM classes. Another neuropeptide, myomodulin, decreased the duration of CM-SB neuron spikes but had no effect on CM-SN spikes. Since the CM neurons appear to mediate a variety of competing behaviors, including feeding, locomotion, and defensive withdrawal, the various neuromodulator actions may contribute to the mechanisms whereby behaviors are selected and modified.

Action Potentials↗

Genomic organization of the FMRFamide gene in Lymnaea: multiple exons encoding novel neuropeptides.

Based on the sequencing of genomic and cDNA clones, we were able to determine that the FMRFamide gene consists of five exons covering at least 20 kb and predict the presence of further novel peptides. The exons are alternatively spliced: exon I (hydrophobic leader sequence) to exon II (tetrapeptides) and exon I to exons III (heptapeptides), IV, and V. A cDNA clone encoding the heptapeptides is described and has also been shown to encode further novel peptides SKPYMRFamide, HDYMRFamide, and SSFPRYamide. Analysis of the right internal parietal nerve using mass spectrometry showed that the novel peptide SKPYMRFamide was cleaved from the precursor. This peptide excites neurons, suggesting a physiological function in the CNS.

Amino Acid Sequence↗

Mutually exclusive expression of alternatively spliced FMRFamide transcripts in identified neuronal systems of the snail Lymnaea.

The FMRFamide gene of the snail Lymnaea encodes tetrapeptides (FMRFamide/FLRFamide) and heptapeptides (GDPFLRFamide/SDPFLRFamide) on separate exons. In situ hybridization probes specific to these exons were used to map the expression of the two exons in identified neuronal systems of the CNS. Analysis of more than 200 preparations showed that cytoplasmic expression of mRNA was exclusively of one type, with individual neurons expressing either the tetrapeptide or heptapeptide exon. Of the approximately 340 neurons expressing the two exons, the majority (80%) expressed the tetrapeptide exon. The tetrapeptide exon was more widespread, occurring in neurons from all 11 ganglia of the CNS. The heptapeptide was mainly confined to two ganglia (visceral and right parietal), with a small number of cells in three other ganglia. Mapping studies combined with dye marking of identified neurons showed the presence of the tetrapeptide exon in several behaviorally important networks: heart motoneurons, whole body withdrawal response motoneurons, and probably penis motoneurons as well as giant identified neurons (LP1, RPD1). The heptapeptides were prominent in two main clusters of cells (Bgp and Fgp) together with a smaller number of tetrapeptide-expressing cells.

Amino Acid Sequence↗

Immunocytochemical evidence for the involvement of an FMRFamide-related peptide in egg production in the flatworm parasite Polystoma nearcticum.

The monogenean flatworm Polystoma nearcticum exhibits reproductive synchrony with its treefrog host, Hyla versicolor, and becomes reproductively active only during the short period of host sexual activity at spawning. In this way, it provides a useful model system for exploring factors that may influence egg production in flatworm parasites. One such factor is the peptidergic innervation of the egg chamber or ootype. By using immunocytochemical techniques, the occurrence and distribution of GYIRFamide-like immunoreactivity, an authentic flatworm FMRFamide-related peptide (FaRP), have been monitored in the cells and fibres innervating the reproductive apparatus of worms collected at different stages of host sexual activity. Serotonin (5-HT) immunoreactivity in the worm was mapped for comparison. Extensive immunostaining for the FaRP and 5-HT was obtained throughout both the central and the peripheral nervous systems of worms, which were recovered from reproductively active frogs. In contrast, the innervation of the ootype of worms that were determined to be sexually inactive, including those recovered from frogs postspawning, showed little or no immunoreactivity for the FaRP; immunostaining for 5-HT in the ootype was unaffected by the reproductive state of the worm. These results indicate that FaRP expression in the neurons of the ootype innervation of P. nearcticum coincides with the parasite's brief period of egg production and, thus, provides evidence that regulatory peptides may be involved in the egg-assembly mechanism in flatworm parasites.

Animals↗

The effects of two FMRFamide related peptides (A-18-F-amide and F-8-F-amide; 'morphine modulating peptides') on the endocrine and exocrine rat pancreas.

The effects of two recently isolated mammalian FMRFamide related peptides (A-18-F-amide and F-8-F-amide) on the encocrine and exocrine rat pancreas were investigated. A-18-F-amide (10, 100, 1000 pM) inhibited concentration dependently glucose (10 mM)- and arginine (10 mM)-induced insulin secretion from the isolated perfused rat pancreas during the first (controls: 100%; 10 pM: 114%; 100 pM: 63%, p less than 0.05; 1000 pM: 31%, p less than 0.05) and the second secretion phase (controls: 100%; 10 pM: 102%; 100 pM: 78%; 1000 pM: 27%, p less than 0.05). The inhibitory actions of A-18-F-amide on pancreatic D-cell secretion were more pronounced during the first than the second phase (first phase: controls: 100%; 10 pM: 95%; 100 pM: 37%, p less than 0.05; 1000 pM: 39%, p less than 0.05%; second phase: controls: 100%; 10 pM: 113%; 100 pM: 72%; 1000 pM: 59%, p less than 0.05). F-8-F-amide (at 1000 pM) inhibited stimulated insulin (controls: 100%; first phase: 26%, p less than 0.05%; second phase: 20%, p less than 0.05) and somatostatin release (controls: 100%; first phase: 14%, p less than 0.05; second phase: 29%, p less than 0.05). Both peptides were without effect on basal and CCK-8-stimulated amylase release from isolated incubated rat pancreatic acini.

Amino Acid Sequence↗

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.

Journal Article↗

Further characterization of Helix FMRFamide receptors: kinetics, tissue distribution, and interactions with the endogenous heptapeptides.

The biphasic binding of 125I-daYFnLRFamide to crude brain membranes of Helix aspersa is due to two discernible sites (high and low affinity) rather than different agonist-induced states. The tissues in the snail that show the greatest specific 125I-daYFnLRFamide binding are the brain, reproductive system, and digestive system. The heart shows moderate binding levels, whereas low values are obtained in the oviduct and retractor muscles. The N-terminal SAR of the Helix heptapeptides (X-DPFLRFamide) indicates that, although the substitution of Leu for Met accounts for some, the dipeptide X-Asp produces most of the loss in potency at FMRFamide receptors in Helix brain.

Amino Acid Sequence↗

Isolation, sequence, and bioactivity of FMRFamide-related peptides from the locust ventral nerve cord.

The ventral nerve cord of the locust, Locusta migratoria, was examined for the presence of FMRFamide-related peptides (FaRPs). RP-HPLC coupled to an RIA specific for extended -RFamides revealed the presence of several FaRPs eluting at different percentages of acetonitrile. The sequences of five of these peptides were determined. Two sequences are identical to the two peptides previously sequenced from brain and retrocerebral complex of Locusta. These two peptides (PDVDHVFLRFamide and ADVGHVFLRFamide) were inhibitory when tested on locust oviduct contractions. The other peptides are novel with sequences of GQERNFLRFamide, AXXRNFIRFamide, and AFIRFamide. The synthesized peptides were stimulatory when tested on locust oviduct contractions, increasing the frequency and amplitude of spontaneous contractions and resulting in a basal contraction.

Amino Acid Sequence↗