Ruthann Nichols

Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides.

An extract of adult Drosophila melanogaster was separated by gel exclusion, ion exchange, and re-versed-phase chromatography. Four peptides, each with an -ArgPheNH2C-terminal sequence, were identified by radioimmunoassay. The primary sequences were determined by Edman degradation and confirmed by mass spectrometry and sequence-specific radioimmunoassay. Three of the peptides are encoded by Drosophila pro-FMRFamide: AspProLysGlnAspPheMetArgPheNH2 (DPKQDFMRFamide), ThrProAlaGluAspPheMetArg-PheNH2 (TPAEDFMRFamide), and SerAspAsn-PheMetArgPheNH2 (SDNFMRFamide). A novel Drosophila peptide ThrAspValAspHisValPheLeuArgPheNH2 (TDVDHVFLRFamide) was also isolated. TDVDHV-FLRFamide is structurally related to peptides isolated from chicken, cockroach, locust, and snail; the cockroach, fruitfly, and locust peptides differ only by the N-terminal amino acid residue. Two Drosophila neural genes, dsk and FMRFamide, are known to encode -Arg-PheNH2-containing peptides; however, neither encodes TDVDHVFLRFamide,- indicating that Drosophila contains another precursor encoding -ArgPheNH2 peptides.

Neural transmitters and a peptide modulate Drosophila heart rate.

Neural messengers affect Drosophila heart rate. Serotonin increases larval, pupal, and adult heart rate. Octopamine and dopamine are inactive in larva, decrease pupal rate, and increase adult heart rate. Acetylcholine and nicotine decrease larval and pupal heart rate, while acetylcholine decreases and nicotine increases adult heart rate. Muscarine decreases pupal heart rate, but is inactive in larva and adult. GABA is inactive in larva and adult, but decreases pupal heart rate. Glutamate is inactive in larva and pupa, but decreases adult heart rate. Proctolin decreases heart rate in all three stages. Caffeine acts only to decrease adult heart rate.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

Isolation and Expression of the Drosophila Drosulfakinin Neural Peptide Gene Product, DSK-I'

The Drosophila drosulfakinin (dsk) gene encodes the cholecystokinin homologues drosulfakinin-I (DSK-I) and drosulfakinin-II (DSK-II). The naturally occurring DSKI peptide was isolated from an extract of adult flies and its sequence determined by automated Edman degradation and sequence-specific radioimmunoassay. The dsk cDNA is expressed during the larval, pupal, and adult stages of development and is an abundant adult head transcript. Sequence-specific DSK antibodies localized DSK expression in the Drosophila larval central nervous system to medial neurosecretory cells and projections that extend from the neurons anteriorly into the brain and posteriorly down the ventral ganglion. The availability of the dsk transcript, sequence-specific DSK antibodies and the application of molecular genetics provide the opportunity to elucidate the role(s) of Drosophila CCK homologues in brain structure and function.

Callitachykinin I and II, two novel myotropic peptides isolated from the blowfly, Calliphora vomitoria, that have resemblances to tachykinins

Two peptides, related to the locust myotropic peptides locustatachykinin I-IV, were isolated from the blowfly Calliphora vomitoria. Whole, frozen flies were used for extraction with acidified methanol. A cockroach hindgut muscle contraction bioassay was used for monitoring fractions during subsequent purification steps. A series of eight different high performance liquid chromatography column systems was required to obtain optically pure peptides. Two peptides were isolated and their sequences determined by Edman degradation and confirmed by mass spectrometry and chemical synthesis as APTAFYGVR-NH2 and GLGNNAFVGVR-NH2. They were named callitachykinin I and II. The peptides have sequence similarities to the locustatachykinins and vertebrate tachykinins. Both callitachykinins were recognized by an antiserum to locustatachykinin I in enzyme-linked immunosorbent assay (ELISA) tests and callitachykinin II was additionally recognized by an antiserum to the vertebrate tachykinin kassinin, suggesting that immunolabeling of blowfly neurons with these antisera is due to neuronal callitachykinins.

Differential Processing of Neuropeptides Influences Drosophila Heart Rate

Peptides that play critical physiological roles are often encoded in precursors that contain several structurally-related gene products. Differential processing of a precursor by cell-specific processing enzymes can yield multiple messengers with diverse distributions and activities. We have reported the isolation of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from adult 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 processing of the FMRFamide polypeptide protein precursor, we generated antisera to distinguish among the structurally-related neuropeptides. Utilizing a triple-label immunofluorescent protocol, we mapped the distribution of the peptides. Each peptide has a unique, non-overlapping cellular expression pattern in neural tissue suggesting that the precursor is differentially processed. In order to identify a biological activity of the peptides, we established an in vivo heart rate assay. SDNFMRFamide decreases heart rate but DPKQDFMRFamide and TPAEDFMRFamide do not, indicating that the N-terminal residues are critical for this activity. SDNFMRFamide immunoreactivity is present in the aorta, implying that SDNFMRFamide acts locally to affect heart rate; DPKQDFMRFamide and TPAEDFMRFamide antisera do not stain cardiac tissue. Our data support the conclusion that Drosophila contains cell-specific proteolytic enzymes to differentially process a polypeptide protein precursor resulting in unique expression patterns of structurally-related, yet functionally distinct neuropeptides.

Drosophila melanogaster flatline encodes a myotropin orthologue to Manduca sexta allatostatin

We identified a Drosophila melanogaster gene encoding a peptide that dramatically decreases spontaneous muscle contractions and, correspondingly, named the peptide flatline (FLT). This gene consisted of 4 exons and was cytologically localized to 32D2-3. Processing of a predicted 122 amino acid precursor would release pEVRYRQCYFNPISCF that differs from Manduca sexta allatostatin (Mas-AST) by one amino acid, Y4-->F4. FLT does not act as an allatostatin. In situ tissue hybridization further suggests FLT is a novel brain-gut peptide and specifically, the measured activity indicates that it is a potent myotropin. Despite its profound myotropic effect, pupae injected with FLT eclosed.

Regulating the activity of a cardioacceleratory peptide

We measured the effect of crustacean cardioactive peptide on Drosophila heart rate in the animal and in a tissue preparation. Crustacean cardioactive peptide increased in vivo basal heart rate 1%, 6%, and 19% and increased in vitro basal heart rate 52%, 25%, and 35% in larvae, pupae, and adults, respectively. In the tissue preparation, the acceleratory period was followed by decreased in vitro heart rates of 42%, 16%, and 13% in larvae, pupae, and adults, respectively. The effects observed in the animal and tissue and in larvae, pupae, and adults suggest that Drosophila crustacean cardioactive peptide cardiac signaling is modulated and developmentally regulated.

Innervation of dromyosuppressin (DMS) immunoreactive processes and effect of DMS and benzethonium chloride on the Phormia regina (Meigen) crop

Antibody to the dipteran myosuppressin peptide, dromyosuppressin, TDVDHVFLRFamide, stained cells and fibers in the brain, optic lobes, subesophageal ganglion, and thoracico‐abdominal ganglion of the blow fly, Phormia regina (Meigen). Dromyosuppressin‐like immunoreactive fibers were detected in the cardiac recurrent nerve, hypocerebral ganglion/corpora cardiaca complex, crop duct, and crop. In order to explore the mechanisms involved in regulating crop movement, we established an in vitro bioassay. The basal rate of crop movement was 50.8 ± 1.5 contractions per minute. Application of 1 μl of saline to the crop did not significantly affect the rate of movement compared to the basal rate (46.1 ± 1.1 contractions per minute, P < 0.05). Application of 1 μl 10‐6 M dromyosuppressin or 1 μl 10‐3 M benzethonium chloride to the crop slowed the rate to 2.2 ± 0.2 and 6.1 ± 0.7 contractions per minute, respectively. Although other data have previously been interpreted to suggest that dipteran crop contractions do not include a neural component, the neuropeptide dromyosuppressin affected P. regina crop motility. Innervation of the crop and crop duct by dromyosuppressin immunoreactive processes that originated in the central nervous system and the effect of dromyosuppressin on crop muscle contractions suggest that dromyosuppressin is released locally to modulate crop contractions and that crop motility is under neural regulation. Myosuppressins isolated from numerous insects have a high degree of structure identity and reduce spontaneous muscle contractions of the hindgut, oviduct, and heart. Benzethonium chloride, previously identified as a myosuppressin agonist on the cockroach hindgut and locust oviduct, mimicked the effect of dromyosuppressin on the crop. This suggests that structural requirements for myosuppressin receptor binding in the cockroach hindgut, locust oviduct, and fruit fly crop are similar. J. Comp. Neurol. 421:136–142, 2000.

SPATIAL AND TEMPORAL IMMUNOCYTOCHEMICAL ANALYSIS OF DROSULFAKININ (DSK) GENE PRODUCTS IN THE DROSOPHILA MELANOGASTER CENTRAL NERVOUS SYSTEM

Abstract.The spatial and temporal distribution of three peptides, DSK I, DSK II, and DSK 0, encoded by the Drosophila melanogaster drosulfakinin (Dsk) gene, have been examined in the central nervous system. DSK I and DSK II have a -RFamide C-terminus and are structurally similar to sulfakinin peptides; in contrast, DSK 0 contains -SFamide and is not structurally similar to sulfakinins. Antisera specificities were determined by the design of the antigens and confirmed by dot blot analysis and preincubation with peptides prior to their use in immunocytochemistry. The distribution of immunoreactivity suggests that all three DSK peptides are processed from the polypeptide precursor and expressed in many of the same cells. Expression was observed at all developmental stages with an increase in the level of staining and the number of immunoreactive cells as development progresses. Cells in the brain lobe, optic lobe, subesophageal ganglion, thoracic ganglia, and the eighth abdominal neuromere contain DSK-immunoreactive materials. Immunoreactive fibers project from some cells and extend into the brain and ventral ganglion with regions of extensive arborization. DSK 0 immunoreactivity provides initial evidence for the presence of a -SFamide peptide in neural tissue. The observed expression of DSK-immunoreactive materials throughout development in numerous cells of the central nervous system suggests that DSK peptides may serve as hormones, modulators, or transmitters involved in several functions.