[Serotonin and neuropeptide FMRFamide in the nervous system of Opisthioglyphe ranae (Trematoda: Plagiorchiidae). an immunocytochemical study].

The presence and localization of the serotoninergic and FMRFamidergic structures in the nervous system of the trematode Opisthioglyphe ranae, the marsh frog intestinal parasite, was studied using immunocytochemistry. The serotonin-immunoreactive nerve cells and fibers were revealed in the head ganglia, circular commissure, longitudinal nerve cords and their connective commissures, as well as around the oral and ventral suckers, oesophagus and genital pore. FMRF-specific immunoreactivity was observed in the head ganglia, longitudinal nerve cords and terminal parts of the reproductive system. The results obtained are discussed in light of the available data on the presence and functional significance of the above-mentioned neurotransmitters in trematodes.

Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus.

Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.

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.

Regulation of insect steroid hormone biosynthesis by innervating peptidergic neurons.

In insects, steroid hormones named ecdysteroids elicit molting and metamorphosis. The prothoracic gland (PG) is a predominant source of ecdysteroids, where their biosynthesis (ecdysteroidogenesis) is regulated by several neuropeptides. Here, we report that FMRFamide-related peptides (FaRPs) regulate ecdysteroidogenesis through direct innervation of the PG in the silkworm Bombyx mori. We purified a previously uncharacterized Bombyx FaRP, DPSFIRFamide, and identified the corresponding Bombyx FMRFamide gene (Bommo-FMRFamide, BRFa), which encodes three additional FaRPs. All BRFa peptides suppressed ecdysteroidogenesis in the PG by reducing cAMP production by means of the receptor for Bommo-myosuppressin, another FaRP we have previously shown to act as a prothoracicostatic factor. BRFa is predominantly expressed in neurosecretory cells of thoracic ganglia, and the neurons in the prothoracic ganglion innervate the PG to supply all four peptides to the gland surface. Electrophysiological recordings during development confirmed the increased firing activity of BRFa neurons in stages with low PG activity and decreased ecdysteroid levels in the hemolymph. To our knowledge, this study provides the first report of peptides controlling ecdysteroidogenesis by direct innervation.

Inter-phyla studies on neuropeptides: the potential for broad-spectrum anthelmintic and/or endectocide discovery.

Flatworm, nematode and arthropod parasites have proven their ability to develop resistance to currently available chemotherapeutics. The heavy reliance on chemotherapy and the ability of target species to develop resistance has prompted the search for novel drug targets. In view of its importance to parasite/pest survival, the neuromusculature of parasitic helminths and pest arthropod species remains an attractive target for the discovery of novel endectocide targets. Exploitation of the neuropeptidergic system in helminths and arthropods has been hampered by a limited understanding of the functional roles of individual peptides and the structure of endogenous targets, such as receptors. Basic research into these systems has the potential to facilitate target characterization and its offshoots (screen development and drug identification). Of particular interest to parasitologists is the fact that selected neuropeptide families are common to metazoan pest species (nematodes, platyhelminths and arthropods) and fulfil specific roles in the modulation of muscle function in each of the three phyla. This article reviews the inter-phyla activity of two peptide families, the FMRFamide-like peptides and allatostatins, on motor function in helminths and arthropods and discusses the potential of neuropeptide signalling as a target system that could uncover novel endectocidal agents.

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon.

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.

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.

Peptidergic control of egg-laying in the cephalopod Sepia officinalis: involvement of FMRFamide and FMRFamide-related peptides.

The peptidergic control of egg-laying was investigated in Sepia officinalis by using a myotropic bioassay. Three myotropic high-performance liquid chromatography fractions were obtained from optic lobe extracts. In the first fraction, FMRFamide (FMRFa) and FLRFa were isolated and sequenced. FMRFa-related peptides then were sought by dotting immunobinding of optic lobes extracts. The four immunoreactive fractions detected revealed the occurrence of FMRFa, FLRFa, FIRFa, and ALSGDAFLRFa predicted by the precursor already cloned from the optic lobes of S. officinalis (J Exp Biol 200:1483-9;1997). These peptides clearly appeared to be involved in the regulation of oocyte transport through the oviduct: the tetrapeptides FMRFa and FLRFa stimulated the contractions, whereas FIRFa and ALSGDAFLRFa lowered the tonus, the frequency, and the amplitude of the contractions. The occurrence of FaRPs in the nervous endings of the accessory sex glands suggested that this peptide family is involved in the regulation of secretory processes of the egg capsule. Indeed, FMRFa modulates the contractions of the main nidamental glands in vitro and, thus, should induce mechanical release of the secretion in vivo during ovulation. These results show that the FaRPs could play an important role in the synchronization of ovulation and egg capsule coating.

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus.

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 microM)-induced contractions, with a threshold for activity of 1 microM. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus.