KRAJNIAK, K.G.*; KLOHR, S.D.: Structure Activity Relationship of the FMRFamide Receptor in the Earthworm Crop-gizzard

Previously we have shown the neuropeptide FMRFamide can increase the rate of spontaneous contractions in the isolated earthworm crop-gizzard. Since other tetrapeptide and heptapepitde FMRFamide related sequences have been found in annelids, we used the Lumbricus terrestris crop-gizzard assay to determine how changes in peptide structure would affect this excitatory response. The crop-gizzard was removed from the animal, attached to a smooth muscle transducer, and challenged with increasing concentrations of neuropeptide. A log-concentration curve was used to determine the neuropeptide EC₅₀ value. The EC₅₀ of FMRFamide was 32 nM, while those of FLRFamide and FTRFamide were 320 nM and 3 μM, respectively. Shortening the peptide to MRFamide increased the EC₅₀ to 3.2 μM. When norleucine (nL) was substituted for methionine (M) in the tripeptide nLRFamide contraction rate actually decreased with an EC₅₀ of 52 nM. When d-amino acids were substituted for the normal l-amino acids the following EC₅₀ values were obtained. For d-FMRFamide it was 32 nM and for FMd-RFamide it was 10 nM, while there were no responses to Fd-MRFamide or FMRd-Famide. Although the EC₅₀ of FMd-RFamide was lower than that of FMRFamide, the maximal effect of FMd-RFamide was only 25 percent that of FMRFamide. The heptapeptide GDPFLRFamide had an EC₅₀ of 310 nM, while there was no response to pQDPFLRFamide. These data suggest that the earthworm crop-gizzard receptor prefers a tetrapeptide sequence with MRFamide in the C-terminal for full tissue response.