Examination of the role of FMRFamide-related peptides in the circadian clock of the cockroach Leucophaea maderae.

The accessory medulla, the circadian clock of the cockroach Leucophaea maderae, is abundant in neuropeptides. Among these neuropeptides are the FMRFamide-related peptides (FaRPs), which generally share the C-terminal RFamide. As a first step toward understanding the functional role of FaRPs in the circadian clock of the cockroach, immunocytochemistry with antisera against various FaRPs, MALDI-TOF mass spectrometry, and injections of two FaRPs combined with running-wheel assays were performed. Prominent FMRFamide-like immunoreactivity was found in maximally four soma clusters associated with the accessory medulla and in most neuropils of the protocerebrum. By MALDI-TOF mass spectrometry, various extended FMRFamides of the cockroach L. maderae were partially identified in thoracic perisympathetic organs, structures known to accumulate extended FMRFamides in insects. By mass match, several of these peptides were also detected in the accessory medulla. Injections of FMRFamide and Pea-FMRFa-7 (DRSDNFIRF-NH(2)) into the vicinity of the accessory medulla caused time-dependent phase-shifts of locomotor activity rhythms at circadian times 8, 18, and 4. Thus, our data suggest a role for the different FaRPs in the control of circadian locomotor activity rhythms in L. maderae.

Presence of two neuropeptides in the fusiform ganglion and reproductive ducts of Octopus vulgaris: FMRFamide and gonadotropin-releasing hormone (GnRH).

We have found evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the central nervous system (CNS) and in the reproductive ducts of both female and male cephalopod Octopus vulgaris. Cell bodies and fibers were immunolocalized in the fusiform ganglion from which the nerves that reach the female and male reproductive ducts arise. FMRFamide-like and cGnRH-I-like immunoreactive nerve endings were present in the oviduct, and in the oviducal gland of the female and in the seminal vesicle of the male. The GnRH-like peptide from the reproductive ducts has been partially characterized by HPLC. The retention time of the Octopus vulgaris GnRH-like peptide was similar to the retention time of cGnRH-I. Based on these observations we suggest that FMRFamide-like and a novel GnRH-like peptide are involved in the control of reproductive ducts of Octopus vulgaris. One possibility is that the peptides affect gamete transport. Another possibility is that they regulate secretory products such as mucus and mucilaginous substances from the oviducal gland and the seminal vesicle. Our data provide further evidence to support the hypothesis of the existence of a central and peripheral peptidergic control of reproduction of Octopus vulgaris.

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.

Neuropeptide precursor processing detected by triple immunolabeling.

Peptides that play critical physiological roles are often encoded in precursors that contain several gene products. Differential processing of a polypeptide precursor by cell-specific proteolytic enzymes can yield multiple messengers with diverse distributions and functions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from Drosophila melanogaster. The peptides are encoded in the FMRFamide gene and have a common C-terminal FMRFamide but different N-terminal extensions. In order to investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish between the structurally related neuropeptides. We established a triple-label immunofluorescence protocol using antisera raised in the same host species and mapped the neural distribution of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide. Each peptide has a unique, nonoverlapping cellular expression pattern, suggesting that the precursor is differentially processed. Thus, our data indicate that D. melanogaster contains cell-specific proteolytic enzymes to cleave a polypeptide protein precursor, resulting in unique expression patterns of neuropeptides.