Molluscan Neuropeptide FMRFamide Research Articles

The effect of the neuropeptide FMRFamide on Aplysia californica siphon motoneurons involves multiple ionic currents that vary seasonally.

The molluscan neuropeptide FMRFamide has a number of inhibitory actions on the sensory neurons and motoneurons mediating the defensive gill and siphon withdrawal reflex pathway of Aplysia californica. Exogenous application of FMRFamide has a biphasic, dual-polarity effect on the majority of LFS siphon motoneurons, causing a transient depolarization followed by a prolonged hyperpolarization. FMRFamide induces this response in LFS neurons by causing an increase in multiple ionic currents, including a transient Na+ current, a slow prolonged Na+ current, a 4-aminopyridine (4-AP)-sensitive K+ current and a 4-AP-insensitive K+ current. We have found that a subset of LFS neurons exhibits an exclusively excitatory, biphasic response to FMRFamide, consisting of a transient depolarization followed by a prolonged depolarization of reduced magnitude. Over a period of 29 months, we consistently observed an increase in the incidence of the exclusively excitatory response during the summer months (June to September). From October to May, we observed an exclusively excitatory response to FMRFamide in 19 % of LFS neurons; yet, in the summer months, 51 % of LFS neurons exhibited this response pattern. We compared the ionic basis of the exclusively excitatory response to FMRFamide with the ionic mechanisms mediating the more frequently observed excitatory/inhibitory response. The exclusively excitatory response involves three of the same ionic components as the more typical excitatory/inhibitory response, including the activation of a transient Na+ current, a slow prolonged Na+ current and a 4-AP-insensitive K+ current. The principal difference between the two response types is that FMRFamide fails to activate a 4-AP-sensitive K+ current in those LFS neurons that exhibit an exclusively excitatory response to the peptide. In addition, LFS neurons with an exclusively excitatory response tend to show a coordinated increase in the magnitude of the inward current component of the FMRFamide response. Together, these changes during the summer months may enable this modulatory peptide to bring LFS neurons to suprathreshold levels of activity for eliciting a siphon withdrawal and should substantially alter the neuromodulatory effects of the peptide.

DEVELOPMENT OF NEURONS EXHIBITING FMRFAMIDE-RELATED IMMUNOREACTIVITY IN THE CENTRAL NERVOUS SYSTEM OF LARVAE OF THE SPIDER CRAB HYAS ARANEUS L. (DECAPODA: MAJIDAE)

ABSTRACT The ontogeny of neuropeptide-expressing neurons during larval development of the crustacean central nervous system is as yet poorly understood. Recently, homologues of the molluscan neuropeptide FMRFamide have been isolated from the central nervous system of adult crustaceans. In order to identify sets of neurons which might express FMRFamide-related neuropeptides and to follow their development, whole mount preparations of the brain and ventral nerve cord of laboratory-reared spider crab larvae (Hyas araneus) were labeled with an antibody against the tetrapeptide FMRFamide. The distribution and morphology of FMRFamide-like immunoreactive neurons, their primary neurites, and neuropils were mapped throughout development of the stages zoea I and zoea 2. Ontogenetic alterations in the staining pattern were recorded and the results compared to the distribution of FMRFamide-like immunoreactivity in other crustaceans.

FMRFamide is endogenous to the Aplysia heart.

The presence of the molluscan neuropeptide FMRFamide was investigated in the heart of the sea hare, Aplysia californica. Immunohistochemical localization and high performance liquid chromatography (HPLC) coupled with radioimmunoassays of HPLC fractions were used to demonstrate the presence of FMRFamide and FLRFamide in the heart. FMRFamide-immunoreactive (FMRFamide-IR) nerve fibers, varicosities, and neuronal somata were observed in whole-mounts of the hearts. The atrium and atrioventricular (AV) valve regions contained significantly higher densities (P < 0.05, ANOVA) of immunoreactive varicosities compared to the ventricle. The high density of FMRFamide-IR varicosities in the atrium and the lack of sensitivity of this region to FMRFamide suggest that the atrium may be a neurohemal organ for the release of FMRFamide. The presence of FMRFamide-IR somata in the Aplysia heart suggests that peripheral neurons may play a role in modifying heart activity, independent of the central nervous system.

FMRFamide produces biphasic modulation of the LFS motor neurons in the neural circuit of the siphon withdrawal reflex of Aplysia by activating Na+ and K+ currents

The molluscan neuropeptide FMRFamide has an inhibitory effect on transmitter release from the presynaptic sensory neurons in the neural circuit for the siphon withdrawal reflex. We have explored whether FMRFamide also acts postsynaptically in motor neurons in this circuit, focusing on the LFS motor neurons. FMRFamide typically produces a biphasic response in LFS neurons: a fast excitatory response followed by a prolonged inhibitory response. We have analyzed these postsynaptic actions and compared them with the mechanism of FMRFamide's inhibition of the presynaptic sensory neurons. The transient excitatory effect of FMRFamide, which desensitizes rapidly, is due to activation of a TTX-insensitive, Na(+)-dependent inward current. The late hyperpolarizing phase of the FMRFamide response results from activation of at least two K+ currents. One component of the hyperpolarizing response is active at rest and at more hyperpolarized membrane potentials, and is blocked by 5 mM 4-aminopyridine, suggesting that it differs from the previously described FMRFamide-modulated K+ currents in the presynaptic sensory neurons. In addition, FMRFamide increases a 4-aminopyridine-insensitive K+ current. Presynaptically, FMRFamide increases K+ conductance, acting via release of arachidonic acid. In the LFS motor neurons, application of arachidonic acid mimicked the prolonged, hyperpolarizing phase of the FMRFamide response; 4-bromophenacyl bromide, an inhibitor of phospholipase A2, selectively blocked this component of the FMRFamide response. Thus, FMRFamide may act in parallel pre- and post-synaptically to inhibit the output of the siphon withdrawal reflex circuit, producing this inhibitory effect via the same second messenger in the sensory neurons and motor neurons, though a number of the K+ currents modulated in these two types of neurons are different.

The possible role of vena cava peptides in regulation of the branchial hearts of Sepia officinalis L. (cephalopoda)

AbstractAn analysis was made of the effects of 5 neuropeptides on the isolated branchial heart of Sepia officinalis. There is evidence to suggest that these 5 neuropeptides occur in the neurosecretory system of the vena cava (NSV system) of cephalopods. YGGFMRFamide and oxytocin caused concentration‐dependent increases in pressure amplitude in the perfused preparations, with their effects on heart rate being negligible. The molluscan neuropeptide FMRFamide hardly had any effect on the branchial heart beat when applied alone, but it counteracted the positive inotropic actions of noradrenaline. FLRFamide and α‐melanotropin had no effect in our system. None of the peptides tested in bioassays on branchial hearts was as efficient as a perfusate obtained from the cephalic vein of Sepia (including the NSV area) that had been stimulated by electrical irritation of the attached visceral nerves. These findings suggest that there may be further neurohormones in the NSV system that are released into the vessel lumen. It is suggested that the branchial hearts of Sepia in vivo are influenced by humoral peptides from the NSV system and that these peptides may act directly on target myocytes or may alter (i.e., potentiate or oppose) the effects of other cardioactive compounds. © 1992 Wiley‐Liss, Inc.

The effects of FMRFamide, 5-hydroxytryptamine and phorbol esters on the heart of the mussel Geukensia demissa.

The ventricle of the mussel Geukensia demissa is inhibited by 5-hydroxytryptamine and excited by the molluscan neuropeptide FMRFamide. Supra-threshold doses of amide result in marked positive chronotropy and inotropy within 5-15 s. 5-Hydroxytryptamine at 10(-8) M produces diastolic arrest within 10 s. A 1-min exposure to FMRFamide (5 x 10(-8) M) results in a small increase in the cytoplasmic levels of adenosine 3',5'-cyclic monophosphate; shorter or longer exposures have no effect. The cAMP content of ventricles incubated in 5 x 10(-8) M 5-hydroxytryptamine for 1 min decreases by 2.3 pmol/mg protein; longer or shorter incubations have no effect. Treatment with forskolin results in 3- or 4-fold increases in adenosine 3',5'-cyclic monophosphate, but forskolin has no effect on the mechanical activity of the ventricle. The levels of inositol monophosphate, inositol 1,4-diphosphate, and inositol 1,4,5-triphosphate in tissues exposed to 5-hydroxytryptamine are not different from levels in control tissues. FMRFamide decreases the levels of these phosphoinositides by 50% or more. Lower concentrations of phorbol 12,13-diacetate (10(-8) to 10(-7) M) and phorbol 12-myristate,13-acetate (10(-6) M) cause positive chronotropy in the isolated ventricle; higher concentrations induce systolic arrest. These results suggest that the effects of 5HT on the ventricle are not mediated by adenosine 3',5'-cyclic monophosphate or inositol 1,4,5-triphosphate. The effects of FMRFamide may involve a decrease in inositol 1,4,5-triphosphate. The effects of amide may involve a decrease in inositol 1,4,5-triphosphate. The response of the ventricles to phorbol esters suggest that protein kinase C may be involved in the regulation of cardiac contractility.

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) can activate a ligand-gated ion channel in Helix neurons

This report presents evidence that the molluscan neuropeptide FMRFamide can directly activate a ligand-gated ion channel in Helix neurones. Using the patch-clamp technique we have observed unitary currents activated by the application of FMRFamide onto outside-out patches. As for the whole-cell response, Na+ ions are the main charge carriers. We conclude that FMRFamide may act as a fast depolarizing neurotransmitter in the Helix nervous system.

Excitation of the clam heart by phorbol esters

1. 1. The isolated ventricles of the clam Mercenaria mercenaria are excited by the phorbol esters phorbol 12,13-diacetate (PDA) and phorbol 12-acetate, 13-myristate (PMA). 2. 2. The frequency of the ventricular contractions was increased by both compounds at concentrations of 10 −9 M; the amplitude of the contractions was increased by 10 −9M PDA, PMA did not produce a consistent inotropic effect. 3. 3. These results suggest that the cardioexcitatory effects of 5-hydroxytryptamine and the molluscan neuropeptide FMRFamide may be mediated, in part, by protein kinase C.

The molluscan neuropeptide FMRFamide stimulates the release of [14C]acetylcholine from isolated ileal synaptosomal preparations of guinea-pig

FMRFamide stimulated, in a dose-dependent manner, the efflux of [14C]acetylcholine (ACh) from isolated synaptosomes of guinea-pig ileum preloaded with labelled choline. Participation of the cholinergic mechanism and/or substance P was ruled out by the finding that antagonists failed to affect the FMRFamide-induced release of ACh. The ACh-releasing action of FMRFamide was negated by the deletion of calcium ion from the bathing medium and it was also abolished by tetrodotoxin. The results obtained suggest that FMRFamide possesses the ability to induce the release of ACh from enteric synaptosomes of guinea-pig.

FMRFamide-like Immunoreactivity in the Nervous System of the StarfishAsterias rubens

The nervous system of the starfish Asterias rubens was subjected to immunocytochemical investigation using antisera raised against the molluscan neuropeptide FMRFamide. Immunoreactivity was detected in the radial nerve cords and the circumoral nerve ring, as well as in the sub-epithelial nerve plexus of the tube foot system. The hyponeural part of the radial cords contained numerous immunoreactive cell bodies. In the ectoneural tissue, immunoreactive cells were present in the epithelium, with cell bodies especially abundant in the lateral parts of the nerve, close to the site of emergence of the innervation to the tube feet. The sub-epithelial nerve plexus of the tube feet contained immunoreactive fibers that were continuous with an extensive system of ectoneural immunoreactive fibers in the radial nerve cords. Immunoreactive fibers were particularly evident in the regenerating radial nerves of previously sectioned arms.

Identification by radioimmunoassay and HPLC of morphine modulatory peptide-immunoreactivity in rat spinal cord and brain

Two so-called morphine modulatory peptides, an octapeptide and an octadecapeptide, have recently been isolated from bovine spinal cord. We have raised antibodies to the octapeptide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2: FF-8), which in radioimmunoassay react with peptides terminating in Arg-Phe-NH 2. This dipeptide is common to both the morphine modulatory peptides and the molluscan neuropeptide FMRF amide. The distribution and molecular forms of immunoreactive peptides were examined in the rat central nervous system and gastrointestinal tract. Highest concentrations of FF-8-like immunoreactivity were found in the dorsal spinal cord, brain stem and hypothalamus. The immunoreactive material in central nervous system extracts was resolved by reversed phase HPLC into three peaks of activity, the two largest peaks eluted in similar positions to the standard octapeptide and octadecapeptide. It appears that previously observed FMRF amide-like immunoreactivity in the rat central nervous system corresponds to peptides immunochemically and chromatographically similar to the two bovine spinal cord peptides.

Potentiating effects of some invertebrate neuropeptides on twitch contraction of the radula muscles of a mollusc, Rapana thomasiana

1. 1. In the radula protractor of the prosobranch mollusc Rapana thomasiana, the molluscan neuropeptide FMRFamide (≧ 10 −9 M) potentiated twitch contractions in response to a train of shortduration (0.5–1.0 msec) pulses of stimulation, but in the radula retractor, the peptide did not affect the contractions. 2. 2. FLRFamide (≧ 10 −10 M) , a FMRFamide-related neuropeptide, and the crustacean red pigment concentrating hormone (≧ 10 −8 M) potentiated twitch contractions of the retractor evoked by the same kind of stimulation, whereas they did not markedly change the evoked contractions of the protractor. 3. 3. In both of the muscles, these peptides did not markedly change the contractions in response to acetylcholine and glutamate, which have been suggested to be the principal excitatory neurotransmitters in the protractor and retractor, respectively. 4. 4. In the presence of the acetylcholine-antagonist propantheline (10 −5 M) or high Mg 2+ (80 mM Mg 2+), FMRFamide hardly changed twitch contractions of the protractor in response to a train of long-duration (3–5 msec) pulses of stimulation. 5. 5. In the presence of high Mg 2+, FLRFamide did not markedly change twitch contractions of the retractor in response to the same kind of stimulation, and the crustacean peptide hardly changed the contractions. 6. 6. These results suggest that the peptides act on the presynaptic sites in the protractor or retractor to increase release of excitatory neurotransmitter.

EPITHELIAL WATER PERMEABILITY IN THE EURYHALINE MUSSEL GEUKENSIA DEMISSA: DECREASE IN RESPONSE TO HYPOOSMOTIC MEDIA AND HORMONAL MODULATION.

The diffusional water permeability of isolated mantles from the mussel Geukensia demissa was reduced by incubation ofthe tissues in hypoosmotie media. The permeability of mantles from 1000 mOsm seawater (SW)-accimated animals was 6 x 10-5 cm/s. A four-hour incubation in 500 mOsm SW or 250 mOsm SW reduced the water permeability by 2 x l0-5 cm/s and 4 x 10-5 cm/s, respectively. A half-hour exposure to the hypoosmotic medium was sufficient to induce the decrease in permeability. The water permeability of mantles incubated in isosmotic SW containing acetone extracts of ganglia from 1000 mOsm SW-acclimated mussels or of mantle from 500 mOsm SW-acclimated mussels was significantly reduced. Extracts of gill had no effect. Ovine prolactin (50 mg/ml) decreased the water permeability of mantles in isosmotic seawater. Cortisol (10-4 M), arginine vasopressin (10-6 M), and the molluscan neuropeptide FMRFamide (10-6 M) had no effect. These results show that the epithelial water permeability of euryhaline bivalves varies with changes in the ambient salinity, and that these permeability changes may be modulated by factors of neural origin.

Peptide Synthesis in Aqueous Solution. I. Application of p-Dialkylsulfoniophenols as a Water-Soluble Coupling Reagent

Abstract (p-Hydroxyphenyl)dimethylsulfonium methyl sulfate(HODMSP·MeSO4−) was found to be an excellent coupling reagent having a water-soluble property and a high reactivity; it worked as satisfactory as usual active esters in regard to the reactivity, product purity, and racemization. The marked advantage of the HODMSP·MeSO4− active ester method was the fact that bifunctional residues such as Arg, Lys, Cys, and Tyr could be selectively acylated when the pH of the reaction mixture was controlled. The molluscan neuropeptide FMRFamide(Phe–Met–Arg–Phe–NH2) was synthesized by this new active ester method for an evaluation of this method to further applications involving peptide syntheses.

Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain

A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.

The neuropeptide FMRF-amide decreases both the Ca2+ conductance and a cyclic 3',5'-adenosine monophosphate-dependent K+ conductance in identified molluscan neurons

The molluscan neuropeptide FMRF-amide (10 to 50 microM) decreases the duration of the Ca2+-dependent action potential recorded in the cell body of identified neurons of the snail Helix aspersa (cells D3 and E2). In these neurons, FMRF-amide evokes a decrease of the Ca2+ current resulting from a decrease in Ca2+ conductance. In another single neuron, cell E11, FMRF-amide, besides evoking a decrease of the Ca2+ conductance, induces a decrease of the S-current (Klein, M., J. S. Camardo, and E. R. Kandel (1982) Proc. Natl. Acad Sci. U. S. A. 79: 5713-5717), a K+ current controlled by cyclic AMP. However, in this E11 cell, FMRF-amide also evokes a decrease of the amplitude of the Ca2+ spike plateau. As discussed in the preceding paper (Paupardin-Tritsch, D., L. Colombaioni, P. Deterre, and H. M. Gerschenfeld (1985) J. Neurosci. 5: 2522-2532), it is suggested that these FRMF-amide-induced modulations of ionic conductances involved in the Ca2+-dependent spike recorded in these neuronal somata may intervene in processes of presynaptic inhibition and facilitation.

Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family.

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.

Actions of the molluscan neuropeptide FMRF-amide on neurones in the suboesophageal ganglia of the snail Helix aspersa

1. 1. The effects of the molluscan neuropeptide FMRF-amide were tested on several neurones in the suboesophageal ganglia of the snail Helix aspersa. Almost all neurones tested responded to the peptide, some being hyperpolarized (H response) and others depolarized (D response). 2. 2. The H response is due primarily to an inward potassium current and may be blocked in 20 μM 4-aminopyridine. The hyperpolarizing actions of FMRF-amide and dopamine may be separated by ergometrine which blocks the response to dopamine but not to FMRF-amide. 3. 3. The D response is due mainly to an inward sodium current but this is not blocked by d-tubocurarine, morphine or TTX. It appears to be mediated by a distinct receptor/ionophore as excitation by ACh and 5-HT are both antagonized by d-tubocurarine. 4. 4. The Leu 2-substituted analogue FLRF-amide was found to produce similar H responses to FMRF-amide, but was much less potent at producing D responses. It did, however, produce cross-desensitization of the D response to FMRF-amide, suggesting that it does bind to the FMRF-amide receptor.

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

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

A critical examination of the occurrence of FMRFamide immunoreactivity in the brain of guinea pig and rat

Several reports (cf. Weber et al. (1981) Science 214:1248-1251) have described the extensive occurrence, in rat brain, of material immunologically related to the molluscan neuropeptide FMRFamide. We have reexamined these data in guinea pig and rat, using six different antisera to FMRFamide. Immunoreactive perikarya and fibres were found to be distributed throughout the rodent brain (Table 1). This distribution was roughly similar to that found by Weber and coworkers. However, solid-phase absorption of the antisera with bovine pancreatic polypeptide, which shares an arginine and an amidated aromatic amino acid (RYamide) with FMRFamide, showed that staining in most regions could be due to crossreactivity with bovine pancreatic polypeptide-like (or neuropeptide Y-like) material. Double-labelling experiments with antisera to FMRFamide and bovine pancreatic polypeptide led to the same conclusion. The only structures where no apparent crossreactivity occurred were perikarya and fibres in the nucleus dorsomedialis, ventromedialis, periventricularis and paraventricularis hypothalami and fibres in the area lateralis hypothalami, nucleus parabrachialis, substantia grisea centralis mesencephali, various parts of the formatio reticularis, and spinal cord. Hence only these structures might contain material which is more related to the molluscan tetrapeptide.