FMRFamide-related peptides, partial serotonin depletion, and osmoregulation in Helisoma duryi (Mollusca: Pulmonata).

Serotonergic neurons were studied by specific histological methods, and neurons containing Phe-Met-Arg-Phe-NH2 (FMRFamide)-related heptapeptides were identified with an antiserum specific for these substances in the central nervous system of the freshwater snail Helisoma duryi. Serotonergic neurons and their axons are present in all of the ganglia (paired buccal, cerebral, pedal, pleural, parietal, and single visceral) and major nerves of the central nervous system. Large neurons containing FMRFamide-related peptide immunoreactivity are located in the left parietal and visceral ganglia, whereas a few small neurons are located in the cerebral and pedal ganglia. Both serotonergic and FMRFamide-related peptide-immunoreactive dendrites and varicosities were observed in the kidney. A second antiserum with high affinity for FMRFamide-related heptapeptides was used to measure the levels of the immunoreactive material in various tissues, and such material was found in every tissue analyzed. When snails were exposed to a medium isosmotic to their hemolymph, the levels of immunoreactive FMRFamide-related peptides increased in the hemolymph, central nervous system, mantle, and kidney. Injection of dihydroxytryptamine, which is known to deplete serotonin content in the snail, also reduced the levels of FMRFamide-related-immunoreactive material in the above tissues. Therefore, serotonin may influence the levels of FMRFamide-related peptides in tissues by regulating the rate of their synthesis, axonal transport, or release. Both serotonin and FMRFamide-related peptides could be involved in osmoregulation.

Protein phosphorylation in snail cardiocytes stimulated with molluscan peptide SCPb.

Dissociated muscle cells prepared from hearts of the pulmonate snail Helix aspersa were used to study signal transduction induced by molluscan cardioactive peptides. The effects of SCPb on the cAMP levels of whole hearts and the cell preparation were compared. The cells responded to SCPb with a dose-dependent increase in cAMP that had the same structure-activity relations as seen in the intact tissue. SCPb increased the phosphorylation of a 53 kDa protein in a dose dependent manner; threshold was 10(-9) M. The SCPb-induced phosphorylation was mimicked by forskolin and 8-CPT-cAMP. FMRFamide stimulation had no effect on the phosphorylation of this protein.

The flp-1 propeptide is processed into multiple, highly similar FMRFamide-like peptides in Caenorhabditis elegans.

Previously, we described a gene, flp-1, that encodes seven FMRFamide-like peptides from two alternatively spliced transcripts in the nematode Caenorhabditis elegans. To determine whether all or a subset of the predicted peptides coded for by flp-1 are produced in vivo, we undertook the isolation of FMRFamide-like peptides from C. elegans. Six FLRFamide-containing peptides, all contained within the putative translation products of the flp-1 gene, were isolated from extracts of mixed stage animals. By quantitative PCR analysis of RNA from mixed stage animals, we found that the shorter transcript of flp-1 has a higher level of expression than the longer transcript.

Effects of cardioactive peptides on myocardial cAMP levels in the snail Helix aspersa.

Several cardioactive peptides from the pulmonate snail Helix aspersa were tested for their effects on myocardial cAMP levels, but only the family of small cardioactive peptides (SCPs) were clearly effective. SCP increased cAMP in a dose dependent manner; the time course was phasic. The structure-activity relations of this effect were examined with a set of 3 synthetic analogs having characteristics, at the carboxyterminal, of both the SCPs and FMRFamide-related peptides. The adenylate cyclase activator forskolin mimicked the mechanical effect of SCPs on the heartbeat. We conclude that the effect of SCPs on the Helix heart may be mediated by cAMP.

Evidence for a novel FMRFamide-related heptapeptide in the pulmonate snail Siphonaria pectinata.

Extracts of whole false limpets (Siphonaria pectinata) were analysed to determine their complement of FMRFamide-related peptides. As in other pulmonates, FMRFamide itself was found to account for only a portion of the immunoreactivity; the largest immunoreactive peptide peak eluted during HPLC under acidic conditions at the same position as a peak also found in other pulmonates. This major peak was resolved into two components by HPLC at neutral pH, and one component was identified as the heptapeptide amide, GDPFLRFamide, previously described from Lymnaea. The amino acid composition of the second component indicates that it is also a heptapeptide, but that it has two Asx (aspartic acid or asparaginyl) residues instead of the one found in the previously identified pulmonate heptapeptides.

The brain of Lymnaea contains a family of FMRFamide-like peptides.

Authentic FMRFamide and two FMRFamide-related heptapeptides were purified from the central nervous system of the fresh water snail Lymnaea stagnalis. The sequences of the heptapeptides were determined as: Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide) by modified Edman degradation and enzymatic digestion. Relatively high quantities of the deamidated and therefore non-immunoreactive analogs of these two peptides (SDPFLRF and GDPFLRF) were also found. SDPFLRFamide and GDPFLRFamide were synthesized and were found to be chromatographically and biologically indistinguishable from the natural peptides, confirming the sequences. The log dose-response curves for the chronotropic action of either synthetic peptide on the heart of Lymnaea was very similar to that of FMRFamide. These data indicate that Lymnaea contains a family of FMRFamide-like peptides.

Characterization of myomodulin-related peptides from the pulmonate snail Helix aspersa.

Three myomodulin-related peptides--pQLSMLRLamide, PMSMLRLamide, and SLGMLRLamide--have been purified and sequenced from extracts of whole snails. The level of immunoreactive myomodulin was shown by HPLC and RIA to be widely distributed among 26 different snail tissues, with the highest levels (higher even than those in the central ganglia) occurring in certain male reproductive organs. Synthetic pQLSMLRLamide modified either the spontaneous rhythmic activity or the resting tone of several isolated muscular organs: the aorta, ventricle, upper gut, epiphallus, flagellum, and spermatheca; but the retractor muscles of the pharynx, penis, and tentacle were unaffected.

Characterization and solubilization of the FMRFamide receptor of squid.

The optic lobe of squid (Loligo pealei) contains FMRFamide receptors that can bind an iodinated FMRFamide analog: [125I]-desaminoTyr-Phe- norLeu-Arg-Phe-amide ([125I]-daYFnLRFa). Radioligand binding assays revealed that squid FMRFamide receptors are specific, saturable, high affinity sites (Kd = 0.15 nM) densely concentrated in optic lobe membranes (Bmax = 237 fmole/mg protein). The receptors appeared to be coupled to Gs because guanine nucleotides inhibit receptor binding and the stimulation of adenylate cyclase by FMRFamide is GTP-dependent. Both the binding and cyclase data showed that FMRFamide, but not FMRF-OH, interacts at FMRFamide receptors; thus the C-terminal Arg-Phe-amide is critical for binding. The high binding affinity of FMRFamide (0.4 nM IC50) was specific for FMRFamide-like peptides. The structure-activity relations of many FMRFamide analogs were defined in detail and were nearly identical for both the membrane-bound and detergent-solubilized receptors. We also found that squid optic lobe contains FMRFamide-like reactivity as measured with both a radioimmunoassay and a radioreceptor assay. Moreover, we have sequenced a fragment of genomic DNA that encodes a FMRFamide precursor. Our findings in sum suggest that FMRFamide is a neurotransmitter in squid optic lobe, and that this tissue is a good source from which to purify FMRFamide receptors.

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.

An endogenous SCP-related peptide modulates ciliary beating in the gills of a venerid clam, Mercenaria mercenaria.

The activities of both the lateral and frontal cilia of Mercenaria mercenaria were unaffected, either by the two endogenous SCP-related peptides AMSFYFPRMamide and YFAFPRQamide, or by FMRFamide (all at 10(-6) M). Dopamine (DA) inhibited the lateral cilia; the mean EC50 was 2 x 10(-6) M. The peptide YFAFPRQamide--but neither AMSFYFPRMamide nor FMRFamide--antagonized the inhibition induced by DA; this effect was dependent on both time and dose. At a DA concentration of 5 x 10(-7) M, the effect of YFAFPRQamide appeared within 20 min and became maximal within 40-60 min; the mean EC50 at these times was 4.7 x 10(-11) M. If the concentration of DA was increased to 10(-6) M, the maximal effect of the peptide was delayed to 50 min, and the mean EC50 increased to 1.1 x 10(-7) M. Particle transport by the frontal cilia was inhibited by 5-hydroxytryptamine (5HT); the mean EC50 was 5.7 x 10(-7) M. Again, only YFAFPRQamide had an antagonistic effect on the 5HT-induced inhibition. At a 5HT concentration of 10(-6) M, the effects of YFAFPRQamide did not appear until 45 min; the mean EC50 was 10(-6) M. When radioimmunoassayed with an SCP antiserum, the elution profile of a gill extract overlapped those of the SCP-related peptides that had previously been identified in extracts of whole animals. These data suggest that all three SCP analogs occur in the gill. Immunohistochemistry of the gill, carried out with a monoclonal antibody raised to SCPB, stained many varicose neuronal fibers. Most of these were associated with the gill musculature, but a sparse innervation of the filaments underlying the cilia was also observed. Some fluorescent nerve cell bodies were also seen in the gill tissue. Our results are consistent with the hypothesis that YFAFPRQamide modulates branchial activities--muscular as well as ciliary--that are associated with feeding.

The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current.

The complete sequence of the FMRFamide precursor cDNA from Helix aspersa is reported here. Since the 5' end of this cDNA is identical to that of the precursor that encodes the heptapeptide analogs of FLRFamide, the two transcripts are probably derived by alternative RNA splicing. A novel pentapeptide, Glp-Phe-Tyr-Arg-Phe-NH2 (pQFYRFamide), predicted from the cDNA sequence, was purified from extracts of H. aspersa ganglia and identified by mass spectroscopy. Partial gene sequences for the FMRFamide precursors of two closely related pulmonate species-Cepaea nemoralis and Polydontes acutangula-were also determined and compared with the cDNA sequence of H. aspersa and a partial gene sequence previously determined from H. pomatia. Not only are the FMRFamide-related sequences and proteolytic processing sites conserved, but the linear arrangement of these landmarks is also retained. Synthetic pQFYRFamide has some effects on the isolated heart and on neuronal potassium currents in H. aspersa that are similar to those of FMRFamide, but it does not activate the neuronal FMRFamide-gated sodium channel.

Seven FMRFamide-related and two SCP-related cardioactive peptides from Helix.

Pulmonate snails have a more complex array of cardioexcitatory peptides than other molluscs, and Helix has a more complex array than most other pulmonates. Since a full characterisation of the cardioexcitatory peptides is necessary for an understanding of physiology, we sought to identify the members of two families of such peptides - the small cardioactive peptides (SCPs) and the FMRFamide-related peptides (FaRPs) - from Helix aspersa. We characterised the peaks of immunoreactivity from HPLC both by their elution times and by their molecular weights as determined by fast atom bombardment mass spectrometry (FABms). These two criteria, used in parallel, facilitated our identification of several known peptides: MNYLAFPRMamide, identical to SCPB of Aplysia; two tetrapeptide FaRPs, FRMRamide and FLRFamide; and three hepatapeptide FaRPs, NDPFLRFamide, SDPFLRFamide and pQDPFLRFamide. Of these peptides, only FMRFamide and pQDPFLRFamide have previously been reported from Helix. We also discovered an additional SCP and two novel FaRPs and sequenced them. The SCP is Ser-Gly-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide (SGYLAFPRMamide), and the heptapeptide FaRPs are Asn-Asp-Pro-Tyr-Leu-Arg-Phe-amide (NDPYLRFamide) and Ser-Glu-Pro-Tyr-Leu-Arg-Phe-amide (SEPYLRFamide). When these nine peptides were tested on isolated Helix ventricles, the SCPs were the most potent cardioexcitors, the heptapeptide FaRPs were next, and the tetrapeptides had the least activity.

The Sequences of Five Neuropeptides Isolated from Limulus using Antisera to FMRFamide.

Five neuropeptides were isolated from CNS extracts of the horseshoe crab Limulus polyphemus by high pressure liquid chromatography (HPLC). The peptides were identified by radioimmunoassays (RIAs) based on two antisera raised to FMRFamide-related peptides (FaRPs). The purified peptides were analyzed by automated sequencing and mass spectrometry, and the following sequences were obtained: DEGHKMLYFamide, GHSLLHFamide, PDHHMMYFamide, DHGNMLYFamide, and GGRSPSLRLRFamide. The first four peptides are members of a novel family with virtually no relationship to FMRFamide. GGRSPSLRLRFamide, on the basis of structural similarity, becomes the second member of a class of FaRPs known previously only from a peptide isolated from mosquito heads. At least one member of the novel family (GHSLLHFamide) inhibits the isolated heart of Limulus.

The identification, localization, and pharmacology of FMRFamide-related peptides and SCPB in the penis and crop of the terrestrial slug, Limax maximus.

1. The molluscan neuropeptides FMRFamide, pQDPFLRFamide, and SCPB were tested on the isolated crop and penis of the terrestraial slug, Limax maximus. FMRFamide and pQDPFLRFamide stimulated the penis and inhibited contractions of the crop. In contrast, SCPB either stimulated or relaxed the penis and increased the tone of the crop. 2. Fibers and varicosities containing immunoreactive (ir-) FMRFamide and ir-SCPB were located in the penis and crop. 3. Extracts of penes, crops, ganglia, and whole animals all contained FMRFamide, FLRFamide, SDPFLRFamide, NDPFLRFamide, and pQDPFLRFamide. 4. These results suggest that the FMRFamide-related peptides and SCPB are involved in the regulation of the reproductive and digestive activities of Limax.

Identified Helix Neurons: Mutually Exclusive Expression of the Tetrapeptide and Heptapeptide Members of the FMRFamide Family.

Extracts were prepared from selected neurons taken from the central ganglia of the pulmonate snail Helix aspersa, and parallel radioimmunoassay (RIA) and high pressure liquid chromatography (HPLC) were used to analyze for FMRFamide-related peptides (FaRPs). Some neurons (e.g., the cerebral C3 neuron) contained only the tetrapeptides related to FMRFamide (tetra-FaRPs), whereas two clusters of neurons in the parietal ganglia contain a preponderance of the hepta-FaRPs. Using cloned cDNA probes in molecular hybridization studies, we showed that the C3 neuron (and some other large neurons) contain the mRNA species that encodes the tetra-FaRPs. In contrast, the neurons of the parietal clusters predominantly express the mRNA that encodes the hepta-FaRPs. These results were confirmed by in situ hybridization with antisense RNA on whole mount ganglia. Each type of experiment supports the view that FaRP-containing Helix neurons express either the tetra-FaRPs or the hepta-FaRPs, but not both.

The molluscan neurosecretory peptide FMRFamide: comparative pharmacology and relationship to the enkephalins.

The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) has diverse actions on excitable tissues of molluscs, including hearts, noncardiac muscles, complex organs, and neurons. The intracellular transducing mechanisms are also diverse and are not readily correlated with particular responses. FMRFamide increases cyclic AMP levels concomitant with both cardioexcitation and inhibition, but not with muscle contraction. In the same tissues, the effects of 5-hydroxytryptamine are dissimilar and are always accompanied by a cyclic AMP increase. FMRFamide and acetylcholine cause similar tonic contractions of the Busycon radula protractor muscle and identical catch contractures of the mytilid anterior byssus retractor muscle, but the ionic basis of excitation and the sources of activator calcium for contraction are not the same for the two agonists. A comparative study of structure-activity relations showed that FMRFamide receptors are heterogeneous. Helix aspersa ganglia contain no FMRFamide, but a close analog occurs and has been tentatively identified. Evidence supporting a proposed homology between FMRFamide-like and opioid peptides is summarized. The effects of the amphiactive heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 on the venus clam rectum support this hypothesis.

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.

SCP-Related Peptides From Bivalve Mollusks: Identification, Tissue Distribution, and Actions.

The SCPs3 are a small peptide family, characterized in gastropods, and implicated in the control of the cardiovascular system and the muscles involved in feeding and gut motility. We aimed to determine the manifestation of this peptide family in the class Bivalvia. Acetone extracts of whole bivalves were fractionated by high pressure liquid chromatography (HPLC), and reactive peaks were identified by radioimmunoassay (RIA). After purification, sequencing, and analysis by mass spectroscopy, three peptides were identified in the clam Mercenaria mercenaria: IAMSFYFPRMamide, AMSFYFPRMamide, and YFAFPRQamide4. SCP-related peptides from two other species were also sequenced: APKYFYFPRMamide and SAFYFPRMamide from an oyster, Crassostrea virginica; and AMSFYFPRMamide (identical to one of the clam peptides) from a cockle, Dinocardium robustum. The tissue distribution and pharmacological actions of the clam SCPs were determined in M. mercenaria, as follows. The levels of peptide in extracts of 12 tissues were estimated by RIA. The largest concentrations of SCP occur in the palps and the visceral ganglia; the levels in the cerebral and pedal ganglia, the rectum, intestinal typhlosole, and gills were substantially lower; and the smallest amounts were found in the heart and the style sac typhlosoles. Immunohistochemistry revealed many cell bodies in the periphery of the ganglia and fibers in the neuropil. Immunoreactive, varicose fibers also occur in the typhlosoles of the intestine and style sac, and in the rectum, gill, and palps. The atrioventricular valves, but not the atria or ventricle proper, contain immunoreactive fibers. Synthetic clam SCPs were assayed on the rectum, the typhlosoles of the intestine and style sac, and the ventricle, all isolated in an organ bath. At low to moderate doses, the SCPs relaxed the muscles of the rectum; higher doses had biphasic actions. The muscles of the intestinal and style sac typhlosoles were relaxed, and spontaneous rhythmicity was slowed by the SCPs. Most ventricles were unresponsive. We conclude that the SCPs isolated in bivalves--though distinctive--are true homologs of those in gastropods. Moreover, the bivalve peptides also serve similar roles, controlling feeding and digestion, and perhaps even cardioactivity.

Characterization of Two Novel Neuropeptides From the Sea Cucumber Holothuria glaberrima.

Two peptides were purified from intestinal extracts of a sea cucumber, Holothuria glaberrima, by high pressure liquid chromatography (HPLC). The peptides were detected by a radioimmunoassay (RIA) based on an antiserum raised to the molluscan peptide, pGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide). Automated sequencing and mass spectrometry indicate that the isolated peptides are: Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-NH2 (SGYSVLYFamide). These are the first peptides to have been isolated from a member of the echinoderm class Holothuroidea. The antiserum used in the RIA of the peptides was also employed in localizing immunoreactive nerve cells and fibers in the intestine of H. glaberrima. The immunohistochemical results suggest that these peptides might be responsible for the FMRFamide-like immunoreactivity reported earlier. Sequence similarities between GFSKLYFamide, SGYSVLYFamide, and a pair of peptides previously isolated from starfish lead us to propose that all four molecules are members of a family of peptides acting as neurotransmitters in echinoderms.

The Sequencing, Synthesis, and Biological Actions of an ANP-Like Peptide Isolated from the Brain of the Killifish Fundulus heteroclitus.

We have extracted, purified, and sequenced an ANP-like peptide from the killifish. The peptide was extracted from whole brains with acidic acetone, and the aqueous phase remaining after evaporation of the acetone was subjected directly to HPLC. A pure peak was obtained after three successive HPLC steps. A key part of our purification method was the deliberate oxidation of methionyl residues in the peptide between the second and third HPLC steps. The purified peptide was chemically sequenced, and its molecular weight was determined by fast atom bombardment mass spectrometry (FABms). The peptide is 22 amino acids long and has considerable sequence similarity to the known natriuretic peptides, especially within the disulfide bonded "ring"; but unlike these known peptides it ends immediately after the second half cystine. Though it lacks a C-terminal "tail," the killifish peptide is equipotent to rat ANP in our radioimmunoassay, which employs an antiserum to the rat peptide. Furthermore, this brain peptide is equipotent to eel ANP in relaxing toadfish aortic rings, though both fish peptides are slightly less potent than rat ANP.