Identification and localization of a [Met5]-enkephalin-like peptide in the mollusc, Lymnaea stagnalis

The goal of this study was to determine whether [Met5]-enkephalin, or an analog, is present in identified neurons in the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis. High performance liquid chromatography and radioimmunoassay of CNS tissue homogenates revealed both [Met5]-enkephalin and oxidized [Met5]-enkephalin. NO [Leu5]-enkephalin, [Met5]-enkephalin-Arg6-Phe7 or [Met5]-enkephalin-Arg6-Gly7-Leu8 were detected. Quantification of [Met5]-enkephalin, by radioimmunoassay, revealed that the Lymnaea CNS contains approximately 2.2 fmol/CNS (undigested tissue) and 4.5 fmol/CNS (tissue enzymatically digested with trypsin and carboxypeptidase B). The increased amount of [Met5]-enkephalin following tissue digestion indicates the presence of as yet unidentified extended forms of [Met5]-enkephalin in Lymnaea. Using indirect immunocytochemistry, a [Met5]-enkephalin-like peptide was localized to individual cells and cell clusters within the CNS, as well as to fibers in the atrium of the heart. A neuronal map depicting [Met5]-enkephalin-like immunoreactive cells was produced. Among the immunoreactive neurons were four identified, well-characterized, giant cells: VD1, RPD2, LB1 and RB1. Identifiable [Met5]-enkephalin-like immunoreactive neurons were characterized electrophysiologically and morphologically. Additionally, neurons VD1 and RPD2 were confirmed to be immunoreactive to Lymnaea alpha-peptide. The lack of both cross reactivity and sequence homology between alpha-peptide and [Met5]-enkephalin suggests that a [Met5]-enkephalin-like peptide and alpha-peptide are co-localized within these neurons.