Michael J. Greenberg

Relationships between the FMRFamide-related peptides and other peptide families

The relationships between peptide families are recognized in terms of structural similarity and immunological and biological activity. Most of the currently known FMRFamide-related peptides (FaRPs) of molluscs were tested in a radioimmunoassay (RIA) and in the two standard bioassays for FMRFamide: the radula protractor muscle of the whelk Busycon contrarium, and the isolated heart of the clam Mercenaria mercenaria. Some peptides were also tested on the heart of the snail Helix aspersa. The responses of the different assays to these peptides were generally similar, but substantial diversity precluded an absolute resolution of relationships, even among molluscan FaRPs. Nevertheless, this set of responses does constitute a standard against which to estimate the relative affinities of putative FaRPs from other animal groups. Many of the non-molluscan FaRPs (e.g., the pancreatic polypeptide-related peptides, gastrin/CCK, and the opioid peptides) are relatively inactive on the molluscan assays, but others (e.g., LPLRFamide, a peptide isolated from chicken brain; the opioid receptor-modulating peptides A18Fa and F8Fa; and gamma 1-MSH) were relatively potent. Several arthropod FaRPs have substantial FMRFamide-like sequence similarity and immunoreactivity, and they may be homologous members of the molluscan peptide family. However, those structural and functional aspects of peptide families that transcend phyletic lines probably reflect basic principles of binding between peptides and membrane proteins rather than homology.

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.

There is no horohalinicum

Species abundance declines to a minimum (the Artenminimum) between 5 and 8‰, not only in estuaries, but in all bodies of brackish water. Khlebovich (1968) examined published hydrochemical data for estuaries and concluded that sharp changes in the ionic composition of seawater diluted with fresh water occur at salinities below 5 to 8‰. He further argued that these ionic changes constitute a physico-chemical barrier between marine and freshwater faunas. Kinne (1971) gave the name “horohalinicum” to the segment of the salinity gradient between 8 and 5‰. We have re-examined the data used by Khlebovich (1968) and found that, in fact, while the ionic composition of diluted seawater changesslightly between 8 and 5‰, the changes in ionic ratios below 2‰ are much larger. Thus, the proposed physico-chemical barrier does not exist between 8 and 5‰; it cannot then explain the Artenminimum; and there is no basis for the horohalinicum concept of Kinne (1971). Two ecological explanations for the occurrence of the Artenminimum—a species-area effect and the stability-time hypothesis—are discussed and found to be inconsistent with published data on species distributions in brackish waters. The low species diversity of brackish water may be explained, in part, by two factors: few animals evolve those physiological mechanisms required for life in the variable habitat; and these species, which are very eurytopic, have low rates of speciation.

Osmoregulation and salinity tolerance in two species of bivalve mollusc: Limnoperna fortunei and Mytilopsis leucophaeta

This paper reports on salinity tolerance and osmotic regulation in two bivalve molluscs: Mytilopsis leucophaeta Conrad, an oligohaline species (subclass Heterodonta) and Limnoperna fortunei Dunker, a freshwater species (subclass Pteriomorphia). The range of osmolality tolerated by Mytilopsis is 5–400 mOsm. Limnoperna survives in deionized water and in osmolalities of ≤400 mOsm. Both species are osmotic and ionic conformers in media with concentrations of > 70 mOsm; at lower ambient osmolalities, both are hyperosmotic regulators. The major hemolymph ions are Na+ and Cl−; acclimation to dilute media results in hypercalemia in both species. The amino acid content of Mytilopsis soft tissues is 182 μmol · g−1 dry wt in animals acclimated to 10 mOsm. The tissue levels of amino acids increase to 405 μmol · g−1 in Mytilopsis acclimated to 400 mOsm. The free amino acid content of Limnoperna mantle tissue is 9.6 μmol · g−1 dry wt in animals acclimated to 10 mOsm and increases to 35 μmol · g−1 in animals acclimated to 200 mOsm. The differences in osmoregulatory behavior between the two species appear to be quantitative rather than qualitative, despite the phyletic distance between them.

Cardiovascular effects of intraventricular injection of fmrfamide, met-enkephalin and their common analogues in the rat

The molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide), the mammalian opioid peptide met-enkephalin, and their common analogues, met-enkephalin-Arg6-Phe7 (YGGFMRF) and Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide (YGGFMRFamide), were injected into the lateral ventricle of the rat; the cardiovascular effects were studied. FMRFamide caused a rapid, transient elevation in blood pressure accompanied by a great increase in pulse pressure. These effects were followed by secondary increases in blood and pulse pressures. Met-enkephalin produced an initial reduction in blood pressure which was followed by a gradual increase at the higher of two test doses (300 nmole). Injection of YGGFMRF resulted in a gradual increase in blood pressure. This response resembled that to met-enkephalin. The initial response to YGGFMRFamide was similar to that to FMRFamide: increases in both blood and pulse pressures after injection. However, the secondary effect of YGGFMRFamide, a prolonged reduction in blood pressure, was not produced by FMRFamide. These results suggest that the initial excitatory cardiovascular responses may be due to the presence of the C-terminal amide. All of the cardiovascular effects of injecting these peptides into the lateral ventricle were abolished by pre-treatment with naloxone in a dose that, itself, produced no cardiovascular changes. In conclusion, these peptides seem to act via the naloxone sensitive opiate receptors in the rat brain.

Differential effects of the molluscan neuropeptide FMRFamide and the related met-enkephalin derivative YGGFMRFamide on the Helix tentacle retractor muscle

Abstract 1. Low concentrations (0.5 nM and above) of FMRFamide and YGGFMRFamide induce rhythmic contractions of the Helix tentacle retractor muscle. 2. YGGFMRFamide, however, also exerts a pronounced inhibitory effect at higher concentrations, above about 20 nM. and even at lower concentrations it tended to reduce the background tone of the muscle while at the same time inducing rhythmic contractions. 3. The higher doses of YGGFMRFamide, which did not induce rhythmic contractions opposed rhythmic contractions caused by FMRFamide and also suppressed or abolished ACh-induced contractures. 4. YGGFM (met-enkephalin). YGGFL and arginine-vasotocin were without effect on the muscle at concentrations up to 10 μM. Serotonin added during an ACh contracture induced relaxation; it did not induce contractions at concentrations from 5 nM to 500 μM. 5. High doses of FMRFamide also had some inhibitory effect on the muscle, inducing relaxation during ACh-contractures and producing a reduction in the frequency of rhythmic contractions and a reduction in tone compared with peak excitatory effect.

Actions of adenylyl compounds in invertebrates from several phyla: Evidence for internal purinoceptors

Abstract 1. 1. The actions of adenylyl compounds on the internal structures of representative species from several invertebrate phyla were surveyed. 2. 2. Preparations taken from protostomous species were mostly unresponsive or inconsistently responsive. An exception was the terminal intestine of the spiny lobster Panulirus argus , in which ATP, ADP, AMP and adenosine could all potentiate the excitatory response to electrical field stimulation. 3. 3. Preparations isolated from deuterostomous species responded relatively consistently to the adenylyl compounds. There was evidence for ATP-specific, ATP-selective, adenosine-selective, and non-selective purinoceptors in the echinoderms Asterias forbesi, Thyone briareus , and Lytechinus variegatus , and in the urochordate tunicate Ciona intestinalis . 4. 4. The rank orders of agonist potency in some of the tissues were similar to those in vertebrate systems with p 1 - or P 2 -purinoceptors. However, antagonists of the vertebrate P 1 -purinoceptor were ineffective in the invertebrate tissues, indicating that these invertebrate purinoceptors are distinct from those of vertebrates.

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 localization of FMRFamide in the nervous and somatic tissues of Nereis virens and its effects upon the isolated esophagus

1. The tetrapeptide FMRFamide which is present in extracts of Nereis virens was localized in various nereid tissues immunohistochemically. 2. Immunoreactive FMRFamidergic cells and fibers were found in the supraesophageal (brain) and subesophageal ganglia, as well as in the intersegmental ganglia of the ventral nerve cord. Immunoreactive fibers were present in the neuropile of, and the connectives between, the supraesophageal, subesophageal, and intersegmental ganglia. 3. In the periphery immunoreactive FMRFamidergic fibers and a few cell bodies were observed in the gut. Sparse fibers were also seen in the body wall, parapodia, and cephalic palps. When the antiserum was preabsorbed with FMRFamide, no specific immunoreactivity was detected. 4. The esophagus of Nereis, isolated and suspended in a tissue bath, responded to FMRFamide with a dose-dependent relaxation; threshold was between 30 and 300 nM, and the EC50 was 1.55 +/- 0.60 microM. Benzoquinonium did not modify this response. 5. Thus, FMRFamide seems to be a neurotransmitter in both the central and peripheral nervous system of Nereis virens, and may be involved in the control of esophageal motility.

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.