Karen E. Doble

A NOVEL FMRFamide-RELATED PEPTIDE IN HELIX: pQDPFLRFamide

A novel FMRFamide-like peptide, purified from the ganglia of Helix aspersa, has the amino acid sequence: pyroglutamyl-aspartyl-prolyl-phenylalanyl-leucyl-arginyl-phenylalanine amide (pQDPFLRFamide). Synthetic pQDPFLRFamide was prepared; it is chromatographically and biologically indistinguishable from the natural peptide, confirming the sequence. pQDPFLRFamide is about a hundred times more potent than FMRFamide on the isolated Helix heart, but slightly less potent than FMRFamide on the Busycon radula protractor muscle. Since pQDPFLRFamide occurs in Helix blood at levels sufficient to excite the isolated Helix heart, it may act as a cardioregulatory hormone.

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.

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 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.

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.

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 clam rectum is sensitive to FMRFamide, the enkephalins and their common analogs

Abstract The molluscan neuropeptide, FMRFamide (Phe-Met-Arg-Phe-NH2) and eight enkephalin analogs were tested on isolated rectums from Mercenaria mercenaria and Macrocallista nimbosa . The effective enkephalin analogs relaxed the rectum, while FMRFamide contracted it. The relaxations produced by the active enkephalins were qualitatively and quantitatively similar, with one exception. The exceptional analog, YGG-FMRFamide, includes the active sequences of both FMRFamide and the enkephalins and could mimic the effects of FMRFamide, the enkephalins, or both, depending on the dose and preparation. When it acted as a contracture agent, YGG-FMRFamide was equal in activity to FMRFamide. But, as a relaxing agent, it was more potent than any of the enkephalin analogs. Attempts to modify the enkephalin response with naloxone or des-Tyr-Met-enkephalin were unsuccessful, and the effect of [D-Ala2]-Met-enkephalin was somewhat smaller, rather than larger, than that of Met-enkephalin. In conclusion, the isolated clam rectum is sensitive to both FMRFamide and the enkephalins. Specific receptors for both peptides are present, and they do not interact. The opioid receptor most resembles the vertebrate δ-receptor.

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.

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.

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.