IDENTIFICATION, DISTRIBUTION AND PHYSIOLOGICAL-ACTIVITY OF 3 NOVEL NEUROPEPTIDES OF LYMNAEA - EFLRLAMIDE AND PQFYRLAMIDE ENCODED BY THE FMRFAMIDE GENE, AND A RELATED PEPTIDE

We are interested in analysing the detailed modulation of defined neur onal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical an alysis, the pentapeptides EFLRIamide and QFYRIamide. These peptides ma y form part of a new family of peptides sharing the sequence motif -FX RIamide. In this paper we adopt a novel approach to first identify and characterize -FXRIamide-like peptides in extracts from the central ne rvous system of Lymnaea. By a combination of high-performance liquid c hromatography (HPLC) and continuous-flow fast atom bombardment mass sp ectrometry, we identify three novel peptides: EFLRIamide, pQFYRIamide and pQFLRIamide. The first two are those predicted in exon II of the F MRFamide locus whereas the last is, interestingly, a product which can not be derived from post-translational modification of the predicted p eptides but must be encoded by as yet unidentified nucleotide sequence s. A specific antibody raised to EFLRIamide, and immunoreactive to all three peptides, revealed EFLRIamide-like expression throughout the ce ntral nervous system in the same cells where exon II is transcribed an d the peptide SEEPLY (a post-translational product of exon II) was loc alized. Additional cells, however, were also identified. Immunoreactiv ity was mapped in a number of identified neurons in the central nervou s system, including two heart cardioexcitatory motoneurons, the E(he) cells (E heart exciters of the visceral ganglion) and penial motoneuro ns in the right cerebral ganglion. The peripheral tissues (heart and p enial complex) that these respective classes of neurons innervate also exhibited EFLRIamide immunoreactivity. The central and peripheral loc alization of EFLRIamide-like immunoreactivity suggested that EFLRIamid e/pQFYRIamide may have an important physiological role in both these p eripheral systems as well as in the central nervous system, This was c onfirmed by physiological experiments that showed that EFLRIamide and pQFYRIamide inhibited many central neurons and in particular the Bgp n eurons in the right parietal ganglion. EFLRIamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory respons e was followed by a long-lasting increase in the rate of beating. Take n together with earlier work, this study now completes the analysis an d localization of the full set of post-translational products of the F MRFamide precursor in Lymnaea and supplies further evidence towards th e characterization of the physiological systems which such peptides ma y modulate in concert.