Actions of FMRFamide-related peptides on the gCa2+ of the C1 neuron in Helix aspersa.

The endogenous neuropeptides FMRFamide and FLRFamide (tetrapeptides) reversibly reduced a voltage-activated calcium current in the C1 neuron of Helix aspersa by an average of 20%. Two structurally related heptapeptides, pQDPFLRFamide and pQDPFLRIamide, both derived from another precursor protein in this species, did not reduce the current at all.

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.

Costorage and corelease of modulatory peptide cotransmitters with partially antagonistic actions on the accessory radula closer muscle of Aplysia californica.

Many neurons that contain a classical neurotransmitter also contain modulatory peptides, but it has been difficult to establish unequivocally that these peptides are functional cotransmitters. Here, we provide evidence for functional cotransmission in a neuromuscular system of Aplysia. Using immunocytochemical techniques, we localize members of two peptide families, the small cardioactive peptides (SCPs) and the buccalins (BUCs), to a single subset of dense-core vesicles in the terminals of the cholinergic motorneuron B15. We describe a new preparation and method for the direct detection of released peptides and show that the SCPs and BUCs are released when neuron B15 is intracellularly stimulated. Consistent with their subcellular localization, the SCPs and BUCs are released in a stoichiometric ratio that is constant across conditions that change the absolute amount of peptides released. Peptide release is calcium-dependent but does not require muscle contractions. Thus, the release cannot be attributed to a displacement of peptides that may be present in the extracellular space. In previous studies, we characterized the physiological firing patterns of neuron B15. Here, we simulate these firing patterns and show that peptide release occurs. Additionally, we find that significant quantities of material are released under behaviorally relevant conditions. We find that concentrations of released peptides in the muscle are in the concentration range in which exogenously applied peptides exert characterized modulatory actions on muscle contractions. Together, our findings provide strong support for the hypothesis that peptides contained in neuron B15 are functional cotransmitters.

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.

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.

Expression of mRNA encoding FMRFamide-related peptides (FaRPs) in the nervous system of Helix aspersa.

The FMRFamide-related peptides (FaRPs) of Helix fall into two groups with often different pharmacological effects: the tetrapeptides FMRFamide and FLRFamide (tetraFaRPs) and the heptapeptides, which have the general structure XDP(F or Y)LRFamide (heptaFaRPs). Previously, we have shown that each group of FaRPs is encoded within a separate type of cDNA clone, a situation which corresponds to two distinct mRNA species existing in the CNS of Helix. Here, we report on the expression patterns of the two FaRP mRNAs both through embryo-genesis and in the fully differentiated regions of the adult nervous system. The levels and locations of FaRP mRNAs were studied by molecular and in situ hybridization using antisense riboprobes. The onset of expression of FaRP mRNAs occurs in Helix embryos about half-way between egg laying and hatching. First detection of the FaRPs themselves occurs about 2 days later. In embryos, as in the adult CNS, the heptaFaRP mRNA is at least five times more abundant than the tetraFaRP mRNA. In adults, the tetraFaRP mRNA is located primarily in the cerebral ganglia, most obviously in the C3 neuron, but also in a crescent-shaped cluster of small neurons lying anterior to C3. Occasional neurons expressing the tetraFaRP mRNA are detected in the parietal ganglia, but these have not yet been mapped. In contrast, the heptaFaRP mRNA is expressed almost exclusively in the parietal ganglia: in large clusters of about 100 neurons lying near to the anterior surface. The most interesting aspect of FaRP mRNAs is that their expression is not only exclusive to a relatively small number of specified neurons, but that expression appears to be mutually exclusive, that is, a particular neuron expresses only the mRNA for tetra-FaRPs or heptaFaRPs, never both. These results are discussed in relation to what we now know about the structure of the individual mRNA molecules.

Cardiac regulation by endogenous small cardioactive peptides and FMRFamide-related peptides in the snail Helix aspersa.

The putative hepatopeptide pQDPFLRIamide, previously known only from its appearance in a cDNA clone from Helix aspersa, was isolated from circumoesophageal ganglia extracts and sequenced. Extracts of several tissues were fractionated by high performance liquid chromatography and the fractions analysed by radioimmunoassay (RIA). The results indicate that ten cardioactive peptides, FMRFamide, FLRFamide, six FMRFamide-related heptapeptides and two nonapeptide analogues of the small cardioactive peptides (SCPs), are present in the circumoesophageal ganglia (brain), the visceral nerve trunk (from which the cardiac nerve branches) and the aorta. The heart contains the two tetrapeptides, FMRFamide and FLRFamide, and the SCPs, but the heptapeptides were completely undetectable in this organ. The levels of tetrapeptide were high enough to allow their calcium-dependent release from the heart to be demonstrated. Immunohistochemistry revealed a diffuse SCP and FMRFamidergic innervation distributed throughout the heart. These data support the idea that, although the ten peptides are probably acting as neurotransmitters throughout most of the cardiovascular system, the heptapeptides probably also have a neurohormonal rôle on the Helix aspersa heart itself. The binding affinities of the various antisera used in these studies were examined in competitive RIAs, in non-competitive dot-blot assays or in both.

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.

The economic implications of bioengineered mastitis control.

This paper estimates the cost of mastitis for the New York dairy sector. The average cost is found to be $125 per cow from reduced milk production, treatment, and increased culling. At the 1988 cow inventory, this translates to approximately $100 million annually for the entire dairy farm sector. When quality and production losses for the processing sector are added, the cost to the New York industry alone is nearly $150 million annually. Two promising new treatments, a bacteriocin and a vaccine, are evaluated. Both have shown effectiveness in preliminary trials against Staphylococcus aureus. Assuming that further development will allow the treatments to be effective against the major bacterial sources of mastitis infections, the treatments are projected to increase the annual income of the New York dairy industry by $18.8 to $39.7 million. The bacteriocin could replace antibiotic usage, a desirable goal in the opinion of many, and the vaccine promises to immunize cows against mastitis very effectively.

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.

Technology and the family farm.

It is one thing for innovation in agricultural practice to be implemented thoughtlessly; quite another for it to be opposed on principle. The introduction of bovine growth hormone is a case in point.