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 neuronal 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 analysis, the pentapeptides EFLRIamide and QFYRIamide. These peptides may form part of a new family of peptides sharing the sequence motif -FXRIamide. In this paper we adopt a novel approach to first identify and characterize -FXRIamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRIamide, pQFYRIamide and pQFLRIamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRIamide, and immunoreactive to all three peptides, revealed EFLRIamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardioexcitatory motoneurons, the E(he) cells (E heart exciters of the visceral ganglion) and penial motoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that these respective classes of neurons innervate also exhibited EFLRIamide immunoreactivity. The central and peripheral localization of EFLRIamide-like immunoreactivity suggested that EFLRIamide/pQFYRIamide may have an important physiological role in both these peripheral systems as well as in the central nervous system, This was confirmed by physiological experiments that showed that EFLRIamide and pQFYRIamide inhibited many central neurons and in particular the Bgp neurons in the right parietal ganglion. EFLRIamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.