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Molecular properties of various snail peptides from brain and gut.

Attention is focused on the similarities in primary structure of the egg-laying neurohormone of the pulmonate Lymnaea stagnalis and of the opisthobranch Aplysia californica which both consist of 36 amino acid residues. FMRFamide-like peptides have now been isolated and sequenced from six molluscan species. Besides FMRFamide, two closely related peptides were isolated from the central nervous system of L. stagnalis and sequenced. This indicates that a family of FMRFamide-like peptides exist not only in the molluscs, but also within one species. A molluscan growth hormone, isolated from the brain of L. stagnalis, has been characterized. This small peptide hormone stimulates in vitro a receptor-adenylate cyclase system of mantle edge cells and in vivo the Ca2+-incorporation in the shell edge. The biochemical characterization of three vertebrate-like peptides of L. stagnalis, resembling oxytocin, Arg-vasopressin, and insulin, confirms the immunological findings that gastropods contain peptides which are structurally closely related to mammalian peptides.

Adenylyl Cyclases↗

Effect of serotonin and neuropeptides on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus.

The effect of serotonin, FMRFamide and the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. The amine and the cardioactive peptide stimulated the enzyme, although with different potencies, in all tissues studied and, when tested in combination, an additive activation was obtained. FMRFamide induced differential effects in the various targets: marked stimulation of adenylate cyclase, additive to that provoked by serotonin or SCPB, in salivary glands; inhibition of the enzyme, both alone and in combination with the other neuromediators, in the nervous tissue; whereas no influence was found in adenylate cyclase activity in the buccal mass. In the last of these tissues, the peptide might act through an intracellular second messenger other than cyclic AMP. The responsiveness of adenylate cyclase to these neuromediators in all the central ganglia suggested that they can exert an important role as neurotransmitters and/or neuromodulators in the central nervous system of the snail. Moreover, in the light of the differential sensitivity of adenylate cyclase in the salivary glands and buccal mass, we suggest that serotonin, FMRFamide and SCPB modulate the feeding behaviour of P. corneus in a complex way.

Adenylyl Cyclases↗

Modulation of growth cone calcium current is mediated by a PTX-sensitive G protein.

Growth cones of isolated neurons B5 of Helisoma were voltage clamped in the whole-cell configuration. Depolarization of growth cones to -20 mV or greater activated a high-voltage-activated (HVA) calcium current. Addition of the neuropeptide FMRFamide (1 microM), which causes a presynaptic inhibition of synaptic transmission, reversibly reduced the calcium current magnitude. This inhibitory effect is mediated by a pertussis toxin (PTX)-sensitive G protein. Dialysis with the non-hydrolyzable GTP analogs GTP gamma S and Gpp(NH)p caused FMRFamide's effect to become irreversible. Dialysis with GDP beta S or preincubation with PTX prevented FMRFamide from reducing the calcium current. Thus, one role of growth cone G proteins is to modulate ion channels in growth cone membrane which in turn may control growth cone motility.

Animals↗

Isolation and characterization of a Drosophila neuropeptide gene.

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.

Amino Acid Sequence↗

Products of heme-catalyzed transformation of the arachidonate derivative 12-HPETE open S-type K+ channels in Aplysia.

In Aplysia mechanosensory neurons, the neuropeptide FMRFamide increases the opening of the background S-K+ channel. This action is mediated by activation of arachidonic acid metabolism. Arachidonic acid in Aplysia nervous tissue is transformed through the 12-lipoxygenase pathway to 12-HPETE, which undergoes further metabolism. In intact sensory cells, 12-HPETE simulates the FMRFamide response, raising the question of whether 12-HPETE is the messenger molecule ultimately acting on the S-K+ channel. Here we show that in cell-free (inside-out) patches from sensory cells, 12-HPETE fails to modulate the S-K+ channel, but in the presence of hematin (which catalyzes 12-HPETE metabolism), it triggers sharp increases in the channel opening probability. We also found that SKF-525A, an inhibitor of the cytochrome P450, reduces the response to FMRFamide, arachidonic acid, and 12-HPETE in intact cells. We conclude that a heme-catalyzed transformation of 12-HPETE is necessary and sufficient to promote the opening of the S-K+ channel and a heme-containing enzyme such as cytochrome P450 might play this key role.

Animals↗

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in aplysia sensory neurons.

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca2+ concentration ([Ca2+]i). 5-HT caused a 50% increase in the transient rise in [Ca2+]i in response to action potentials, whereas FMRFamide decreased the [Ca2+]i transient by 40%. Neither transmitter altered the resting [Ca2+]i, the time course of recovery of the [Ca2+]i transient, or the [Ca2+]i transients produced by intracellular injection of CaCl2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca2+]i transient are due to changes in Ca2+ influx and not in intracellular Ca2+ homeostasis.

Action Potentials↗

Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition.

FMRFamide evokes both short-term and long-term inhibition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video microscopy, that depression lasting 24 hr of sensorimotor synapses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell varicosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell L7. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in L7, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities.

Animals↗

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors.

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.

Animals↗

Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code.

Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord, the six Tv neurons express the neuropeptide gene FMRFamide. Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain gene apterous. We find that the zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this "code," by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells.

Animals↗

Localization of molluscan cardioexcitatory tetrapeptide in the brain of African Cichlid fish (Haplochromis burtoni) revealed by immunocytochemistry.

The FMRFamide-like immunoreactivity was investigated in the brain of African cichlid fish, Haplochromis burtoni, in which sexual maturation is under social control. In both dominant and subdominant males and females, the FMRFamide immunoreactive (ir) cells were found only in the nucleus olfacto-retinalis and in the nucleus of the midbrain tegmentum. However, several FMRFamide-ir fibers were seen in the olfactory bulb and throughout the entire brain of both male morphs and female fish. As the role of nucleus olfacto-retinalis is well known in chemoreception, these results suggest the involvement of FMFRamide-like peptide in the chemosensory control of reproductive behavior in this species.

Animals↗

Immunocytochemical demonstration of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea).

The localization and distribution of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica at different stages in the development of the adult fluke have been determined by an indirect immunofluorescence technique, using antisera to 19 vertebrate peptides and the invertebrate neuropeptide, FMRFamide. Positive immunoreactivity was obtained with antisera to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP) and FMRFamide. Cell bodies and nerve fibres immunoreactive to the 4 peptides are present in the anterior ganglia and the 3 pairs of longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, immunoreactivity occurs in the nerve plexuses supplying the subtegumental musculature, the oral and ventral suckers, and the muscular lining of the male and female reproductive ducts, including the ootype, uterus, cirrus pouch and gonopore. Cells displaying immunoreactivity to PYY and FMRFamide lie amongst the Mehlis' gland cells that surround the ootype. Processes from these cells extend into the wall of the ootype. One group of PP-immunoreactive cells occurs at the junction of the vitelline and ovovitelline ducts, whilst another group is situated at the entrance to the uterus from the ootype. The results are discussed in relation to the possible roles of the peptides in the neurophysiology and egg production of the fluke.

Animals↗

Immunocytochemical demonstration of neuropeptides in the central nervous system of the roundworm, Ascaris suum (Nematoda: Ascaroidea).

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

Animals↗

Immunocytochemical demonstration of peptidergic and serotoninergic components in the enteric nervous system of the roundworm, Ascaris suum (Nematoda, Ascaroidea).

The localization and distribution of neuropeptides and an indoleamine (serotonin or 5-hydroxytryptamine) in the enteric nervous system (ENS) of the pig roundworm, Ascaris suum, have been determined by the application of an indirect immunofluorescence technique in conjunction with confocal scanning laser microscopy. Whole-mount preparations of pharyngeal, intestinal and rectal regions were screened with antisera to 23 vertebrate peptides, 2 invertebrate peptides and serotonin (= 5-HT). Positive immunoreactivity (IR) was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), FMRFamide, gastrin and serotonin. The only IR observed in the ENS was that evident in the nerve supply to the pharynx and rectal region; no IR was associated with any region of the intestine. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and serotonin. In the pharyngeal component of the ENS, IR was evident in the lateral and dorsal longitudinal pharyngeal nerves, pharyngeal commissures, nerve plexus, and associated nerve cells and fibres. In contrast, the distribution of IR to the PP and gastrin antisera was more restricted and displayed a lower intensity of immunostaining. The other component of the ENS, the rectal enteric system, only yielded immunostaining to FMRFamide. The possible role of neuropeptides and serotonin in the nutritional biology of nematodes is discussed.

Animals↗

A possible role of SchistoFLRFamide in inhibition of adipokinetic hormone release from locust corpora cardiaca.

The distribution and actions of FMRFamide-related peptides (FaRPs) in the corpora cardiaca of the locust Locusta migratoria were studied. Antisera to FMRFamide and SchistoFLRFamide (PDVDHVFLRFamide) label neuronal processes that impinge on glandular cells in the glandular lobe of the corpora cardiaca known to produce adipokinetic hormones. Electron microscopic immunocytochemistry revealed that these FaRP-containing processes form synaptoid contacts with the glandular cells. Approximately 12% of the axon profiles present in the glandular part of the corpus cardiacum contained SchistoFLRFamide-immunoreactive material. Retrograde tracing of the axons in the nervus corporis cardiaci II with Lucifer yellow revealed 25-30 labelled neuronal cell bodies in each lateral part of the protocerebrum. About five of these in each hemisphere reacted with the SchistoFLRFamide-antiserum. Double-labelling immunocytochemistry showed that the FaRP-containing processes in the glandular lobe of the corpora cardiaca are distinct from neuronal processes, reacting with an antiserum to the neuropeptide locustatachykinin. The effect of the decapeptide SchistoFLRFamide and the tetrapeptide FMRFamide on the release of adipokinetic hormone I (AKH I) from the cells in the glandular part of the corpus cardiacum was studied in vitro. Neither the deca- nor the tetrapeptide had any effect on the spontaneous release of AKH I. Release of AKH I induced by the phosphodiesterase inhibitor IBMX, however, was reduced significantly by both peptides. These results point to an involvement of FaRPs as inhibitory modulators in the regulation of the release of adipokinetic hormone from the glandular cells.

1-Methyl-3-isobutylxanthine↗

Regulation of insect steroid hormone biosynthesis by innervating peptidergic neurons.

In insects, steroid hormones named ecdysteroids elicit molting and metamorphosis. The prothoracic gland (PG) is a predominant source of ecdysteroids, where their biosynthesis (ecdysteroidogenesis) is regulated by several neuropeptides. Here, we report that FMRFamide-related peptides (FaRPs) regulate ecdysteroidogenesis through direct innervation of the PG in the silkworm Bombyx mori. We purified a previously uncharacterized Bombyx FaRP, DPSFIRFamide, and identified the corresponding Bombyx FMRFamide gene (Bommo-FMRFamide, BRFa), which encodes three additional FaRPs. All BRFa peptides suppressed ecdysteroidogenesis in the PG by reducing cAMP production by means of the receptor for Bommo-myosuppressin, another FaRP we have previously shown to act as a prothoracicostatic factor. BRFa is predominantly expressed in neurosecretory cells of thoracic ganglia, and the neurons in the prothoracic ganglion innervate the PG to supply all four peptides to the gland surface. Electrophysiological recordings during development confirmed the increased firing activity of BRFa neurons in stages with low PG activity and decreased ecdysteroid levels in the hemolymph. To our knowledge, this study provides the first report of peptides controlling ecdysteroidogenesis by direct innervation.

Amino Acid Sequence↗

Synaptic precedence during synapse formation between reciprocally connected neurons involves transmitter-receptor interactions and AA metabolites.

The cellular mechanisms that determine specificity of synaptic connections between mutually connected neurons in the nervous system have not yet been fully examined in vertebrate and invertebrate species. Here we report on a novel form of synaptic interaction during early stages of synapse formation between reciprocally connected Lymnaea neurons. Specifically, using soma-soma synapses between an identified dopaminergic neuron (also known as the giant dopamine cell), right pedal dorsal 1 (RPeD1), and a FMRFamidergic neuron, visceral dorsal 4 (VD4), we demonstrate that although reciprocal inhibitory synapses re-form between the somata after 24-36 h of pairing, VD4 is, however, the first cell to establish synaptic contacts with RPeD1 (within 12-18 h). We show that VD4 "captures" RPeD1 first as a postsynaptic cell by suppressing its transmitter secretory machinery during early stages of cell-cell pairing. The VD4-induced suppression of transmitter release from RPeD1 was transient, and it required transcription and de novo protein synthesis dependent step in VD4 but not in RPeD1. The VD4-induced effects on RPeD1 were mimicked by a FMRFamide-like peptide. Perturbation of FMRFamide-activated metabolites of the arachidonic acid pathway in RPeD1 not only prevented FMRFamide-induced suppression of transmitter release from the giant dopamine cell but also shifted the synaptic balance in favor of RPeD1, thus making it the first cell to begin synaptic transmission with VD4 within 12-18 h. A single RPeD1 that had developed dopamine secretory capabilities overnight and was subsequently paired with VD4 for 12-18 h was, however, immune to VD4-induced suppression of transmitter release. Under these experimental conditions, both cells developed mutual inhibitory synapses concurrently. Taken together, our data provide evidence for novel synaptic interaction between reciprocally connected neurons and underscore the importance of transmitter-receptor interplay in regulating the timing of synapse formation in the nervous system.

Animals↗

Aminergic and peptidergic modulation of motor function at an identified neuromuscular junction in Helisoma.

Electrophysiological studies suggest that motoneurone B19 in the buccal ganglia of Helisoma makes monosynaptic, cholinergic connections with the supralateral radular tensor (SLT) muscle of the buccal mass. Serotonin (5-HT) and small cardioactive peptide B (SCPB) were found to have peripheral modulatory effects on this motor pathway that are consistent with their previously described central facilitatory effects. Both neurotransmitters, when applied exogenously (10(-6) mol l-1) to isolated buccal ganglion-buccal muscle preparations, potentiated the magnitude of motoneurone B19-evoked muscle contractions (6.3 and 2.7 times, respectively) without affecting excitatory junctional potential (EJP) amplitudes. When applied to single dissociated SLT muscle fibres in cell culture, these modulators had similar effects on acetylcholine (ACh)-evoked muscle fibre shortening, demonstrating that these neuromodulators exert direct actions on the muscle cells. The cardioactive peptide FMRFamide (10(-6) mol l-1), although slightly potentiating muscle contractions in reduced neuromuscular preparations, significantly decreased both ACh-evoked muscle fibre shortening and depolarizing potentials in cultured SLT muscle cells. The differential effects of FMRFamide may, in part, be due to the elimination of interactive effects between multiple neurotransmitters that might exist in semi-intact preparations and in vivo. These results demonstrate that 5-HT, SCPB and FMRFamide in Helisoma can directly modulate the peripheral muscle targets of buccal motoneurones involved in the generation of cyclical feeding behaviour.

Acetylcholine↗

The first peptide-gated ion channel.

Patch-clamp experiments on the C2 neurone of Helix aspersa have shown that the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) directly gates a Na+ channel. The channel is amiloride-sensitive. Activation of this channel is responsible for the fast excitatory action of the peptide. Using primers based on amiloride-sensitive epithelial Na+ channels, a complete cDNA sequence (FaNaCh) was cloned and sequenced from a Helix library. The sequence is predicted to have just two membrane-spanning regions and a large extracellular loop. When expressed in Xenopus laevis oocytes, the channel responded to FMRFamide. Taken together, these data provide the first evidence for a peptide-gated ion channel. Comparison of the properties of the expressed FaNaCh with the native neuronal channel show small differences in the sensitivities to some drugs and in channel conductance. It is not yet clear whether the native channel is a homo-oligomer or comprises other subunits. The peptide FKRFamide is an effective antagonist of FMRFamide on the expressed and neuronal channels. Nucleotide sequences encoding similar channel proteins occur in neurones of species as dissimilar as man and Caenorhabditis elegans. Some channels are thought to be associated with mechano-sensation, at least one is a proton-gated channel and others may also be ligand-gated channels.

Animals↗