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.     

THE EFFECTS OF THREE DROSOPHILA MELANOGASTER MYOTROPINS ON THE FREQUENCY OF FOREGUT CONTRACTIONS DIFFER

Myotropic peptides can be grouped into different families based on their structure. Three Drosophila melanogaster myotropin families are represented by TDVDHVFLRFamide, dromyosuppressin (DMS), pEVRYRQCYFNPISCF, an allatostatin C-type peptide named flatline (FLT), and SDNFMRfamide, a FMRFamide, a FMRFamide-containing peptide. The structures of DMS, FLT, and SDNFMRFamide differ and each peptide is encoded by a different gene. In addition, the spatial and temporal distributions of DMS, FLT, and SDNFMRFamide are dissimilar. DMS, FLT, and SDNFMRFamide each decreases heart rate; however, their effects are profoundly different. Likewise, the effects of these three myotropins on the frequency of the spontaneous contractions of the crop, an anterior portion of the foregut, differ. DMS stops crop movement without recovery for at least 10-min period after applying the peptide, FLT significantly decreases the frequency of spontaneous contractions, but its effect partially reverses within a few minutes after applying the peptide, and SDNFMRFamide only slightly decreased crop motility, an effect that was not significantly different from the effect of saline. The differences in the structures, distributions, and activities of DMS, FLT, and SDNFMRFamide suggest their synthesis and release are under different sensory inputs and regulatory mechanisms, and that roles in affecting the frequency of crop contractions differ.     

Immunocytochemical demonstration of neuropeptides in the peripheral nervous system of the roundwormAscaris suum (Nematoda,ascaroidea)

The localisation and distribution of neuropeptides in the peripheral nervous system of the pig roundwormAscaris suum have been determined by an indirect immunofluoresence technique in conjunction with confocal microscopy. Of the 31 antisera tested, immunostaining was obtained only with antisera to peptide YY (PYY), pancreatic polypeptide (PP) and FMRFamide. Immunostaining for PYY and FMRFamide was evident in the amphidial and papillary ganglia associated with the anterior nerve ring and in the nerves from these ganglia that terminated in sensory receptors within the buccal lips of the parasite. The only peptide immunoreactivity (IR) observed in the reproductive system of either sex was that evident in the nerve supply to the distal region of the vagina in the female worm. It took the form of a well-developed plexus of parallel nerve fibres, cross-connectives and looped commissures. The nerve net diminished in the more proximal region of the vagina. PP-IR was less intense than that for PYY and FMRFamide and was more restricted in distribution, being confined to a small number of nerve fibres in the nerve supply to the vagina; it did not occur in the nerves supplying the anterior sensory receptors. The possible roles of neuropeptides in the sensory and reproductive biology of nematodes are discussed.     

Neuropeptide F-immunoreactivity in the monogenean parasiteDiclidophora merlangi

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite,Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1–39) or to the C-terminal decapeptide (residues 30–39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide inD. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.     

The effects of three Drosophila melanogaster myotropins on the frequency of foregut contractions differ

Myotropic peptides can be grouped into different families based on their structure. Three Drosophila melanogaster myotropin families are represented by TDVDHVFLRFamide, dromyosuppressin (DMS), pEVRYRQCYFNPISCF, an allatostatin C-type peptide named flatline (FLT), and SDNFMRFamide, a FMRFamide-containing peptide. The structures of DMS, FLT, and SDNFMRFamide differ and each peptide is encoded by a different gene. In addition, the spatial and temporal distributions of DMS, FLT, and SDNFMRFamide are dissimilar. DMS, FLT, and SDNFMRFamide each decreases heart rate; however, their effects are profoundly different. Likewise, the effects of these three myotropins on the frequency of the spontaneous contractions of the crop, an anterior portion of the foregut, differ. DMS stops crop movement without recovery for at least a 10-min period after applying the peptide, FLT significantly decreases the frequency of spontaneous contractions, but its effect partially reverses within a few minutes after applying the peptide, and SDNFMRFamide only slightly decreased crop motility, an effect that was not significantly different from the effect of saline. The differences in the structures, distributions, and activities of DMS, FLT, and SDNFMRFamide suggest their synthesis and release are under different sensory inputs and regulatory mechanisms, and that roles in affecting the frequency of crop contractions differ.     

Occurrence and distribution of putative neurotransmitters in the frog-lung parasiteHaplometra cylindracea (Trematoda: Digenea)

The localistion and distribution of the cholinergic, serotoninergic and peptidergic components of the nervous system of the frog-lung flukeHaplometra cylindracea have been determined by the application of standard enzyme cytochemical and immunocytochemical techniques to cryostat sections and whole-mount preparations. Cholinesterase activity (ChE), as indicative of acetylcholine, has been demonstrated cytochemically in the CNS and PNS; however, the anterior ganglia were notably unreactive. The occurrence of serotonin was examined by an indirect immunofluorescence technique, and immunoreactivity (IR) was demonstrable in small, paired anterior ganglia and in fine nerve fibres associated with the somatic muscle, cirrus and gonopore. The peptidergic protion of the nervous system was investigated using antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide, and was visualised by both indirect immunofluorescence and confocal scanning laser microscopy. Positive immunostaining occurred with antisera raised against pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP), peptide histidine isoleucine (PHI) and FMRFamide. Immunoreactivity to PP, PYY and FMRFamide was widespread throughout the nervous system and was evident in large, paired anterior ganglia, the dorsal commissure, main nerve tracts and the extensive array of small fibres that constitute the PNS. In contrast, the distribution of nerves immunoreactive to SP and PHI was less apparent, with PHI-IR occurring exclusively within the fibrous neuropile of the ganglia and in fibres of the ventral nerve cord. Results are discussed with respect to the distribution of the various neurochemical elements and their roles as putative neurotransmitters and/or regulatory molecules.     

FMRFamide-activated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene

The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (相似文献   

Coexistence of peptide immunoreactivity in sensory neurons of the cat

The coexistence of the neuropeptides substance P, cholecystokinin, somatostatin and vasoactive intestinal polypeptide in cat sensory neurons has been examined using peroxidase-anti-peroxidase immunocytochemistry. Attempts were also made to locate cells containing bombesin, neurotensin, [Met]enkephalin and [Leu]enkephalin but no immunoreactivity was found when antisera to these peptides was used. Cells in the dorsal root ganglia were studied by cutting 5 microns serial wax sections or 15 microns cryostat sections. Coexistence was established by applying the antiserum to each peptide to serially adjacent 5 microns sections and establishing the presence of peptide-like immunoreactivity in each of 4 different sections through a single cell. Results showed that the distribution and combinations of coexistence of these neuropeptides in the cat is extremely complex; three and sometimes all four antisera showing immunoreactivity with a single cell. About 21% of all ganglion cells contained some immunoreactivity but there were certainly some small cells which did not contain any immunoreactivity. The coexistence of these peptides differed markedly from that previously reported in the rat suggesting that interspecific differences in the neuropeptide content of cells might be much greater than they are for classical neurotransmitters. The results are discussed in relation to the possible role of neuropeptides and the regulation of their production by sensory neurons.     

Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins.

We have employed monospecific antisera to the major glycoproteins (gp52 and gp36) and the major nonglycoprotein (p27) of the mouse mammary tumor virus (MMTV), and we now report the first isolation of an intracellular MMTV precursor polypeptide to p27. The precursor polypeptide to p27 (Pr75) binds to single-stranded DNA (ssDNA) and can be easily separated from the precursor to gp52 and gp36 (gPr75) by ssDNA-Sepharose column chromatography. [³⁵S]Methionine-labeled Pr75 contained tryptic peptides of p27 and p14 of MMTV. Protein p14 has previously been shown to be capable of binding to ssDNA. In contrast, [³⁵S]methionine-labeled gPr75 contained tryptic peptides of only gp52 and gp36, neither of which binds to ssDNA.     

Immunocytochemical demonstration of 5-hydroxytryptamine (serotonin) and vertebrate neuropeptides in the nervous system of excysted cysticercoid larvae of the rat tapeworm,Hymenolepis diminuta (Cestoda,Cyclophyllidea)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and a number of vertebrate neuropeptides in the nervous system of excysted (0–24 h) cysticercoid larvae of Hymenolepis diminuta were determined by an indirect immunofluorescence technique in wholemount preparations. In the central nervous system, cell bodies and nerve fibres immunoreactive to 5-HT are present in the main commissure, lateral and rostellar ganglia, and the longitudinal nerve cords and their connectives. In the peripheral nervous system, immunoreactive nerve fibres occur in a poorly developed nerve plexus within each sucker. Among the vertebrate peptides tested, antisera to pancreatic polypeptide (PP), polypeptide YY (PYY), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP) gave positive results. Immunoreactivity to PP and PYY paralleled that of 5-HT, with greater numbers of cell bodies present in the different locations within the scolex nervous system, and the sucker plexus being more prominent. The number of PP-reactive cells in the lateral ganglia and main lateral, longitudinal nerve cords increased over the 24-h period in culture. Results with antisera of different specificities to PP and PYY suggest that the immunoreactivity may be due to a peptide with closer structural affinity to PYY than to PP. Immunoreactivity to PHI is restricted to the main lateral nerve cords in the body of 0-h worms, extending into the median nerve cords by 12 h and 24 h. Immunoreactivity to GRP became evident after 12 h in culture and was confined to the longitudinal nerve cords, in particular the median nerve cords. The results are discussed in relation to the proposed transmitter and regulatory roles of 5-hydroxytryptamine and the neuropeptides.     

Ultrastructural localisation of FMRFamide- and pancreatic polypeptide-immunoreactivities within the central nervous system of the liver fluke,Fasciola hepatica (Trematoda,Digenea)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMR-Famide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode,Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and giant nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.     

Immunostaining for peptides of the egg-laying hormone/bag cell peptide precursor protein in the head ganglia of Aplysia

Egg-laying hormone and alpha-bag cell peptide are two neuropeptides derived from a common precursor protein in the marine mollusk Aplysia. Previous studies indicate that they are neurotransmitters that co-exist in individual bag cell neurons and most bag cell processes in the abdominal ganglion. In the present investigation we used double-label immunocytochemistry with highly specific antisera to describe their distribution elsewhere in the CNS. We found that a small cluster of cells and their fibers in the pleural ganglion that were previously described as being immunoreactive for egg-laying hormone were also immunoreactive for alpha-bag cell peptide(1-9). A previously described group of small cell bodies in the cerebral ganglion also stained for both peptides. However, the fiber arborizations located near them were immunoreactive for alpha-bag cell peptide(1-9), but not egg-laying hormone. This suggests that there is alternative processing of the precursor protein or differential transport of the peptides from the cell bodies. The specificities of the antibodies indicate that all of the neurons that stain for egg-laying hormone-like peptides in the CNS synthesize peptides derived from the egg-laying hormone/bag cell peptide precursor, rather than peptides derived from other members of the egg-laying hormone gene family. They also suggest that peptides derived from the related A or B precursor proteins are not synthesized in the CNS, or at levels too low to detect. The results are consistent with the proposal that the behavior associated with egg-laying is initiated and controlled by peptide transmitters derived from a single gene and expressed in specific neurons of the CNS.     

Serotonin and neuropeptide immunoreactivities in the intramolluscan stages of three marine trematode parasites

Using an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy, whole-mount preparations of three general of marine trematode larvae,Cryptocotyle lingua, Cercaria emasculans andHimasthla leptosoma, were screened for 5-hydroxytryptamine (5-HT) and selected neuropeptide immunoreactivities (IRs). IRs for pancreatic polypeptide (PP), peptide YY (PYY) and FMRFamide were found in the central nervous systems of the three species of cercariae, immunostaining the paired ganglia and central commissure and the longitudinal nerve cords, with slight differences in both distribution and intensity of IRs being observed for the different antisera used. PP, PYY and FMRFamide IRs were evident in both central and peripheral components of the nervous system in the rediae ofC. lingua. 5-HT IR was confined to the peripheral nervous systems of the cercariae ofC. emasculans and the rediae ofC. lingua, appearing in the form of a network of immunoreactive fibres and associated large cell bodies. A moderate substance P IR was observed in the nervous system of the cercariae ofC. lingua. The patterns of immunostaining described were compared with those obtained using antiserum directed to the C-terminal decapeptide amide of neuropeptide F (NPF), a native parasitic peptide from the cestodeMoniezia expansa. Results demonstrated that serotoninergic and peptidergic components were present in the nervous systems of all of the trematode larvae studied and that some, if not all, of the IR for PP, PYY and FMRFamide was due to the presence of a trematode NPF homologue.     

FMRFamide neuropeptides and acetylcholine synergistically inhibit egg-laying by C. elegans

Egg-laying behavior of the Caenorhabditis elegans hermaphrodite is regulated by G protein signaling pathways. Here we show that the egg laying-defective mutant egl-6(n592) carries an activating mutation in a G protein-coupled receptor that inhibits C. elegans egg-laying motor neurons in a G(o)-dependent manner. Ligands for EGL-6 are Phe-Met-Arg-Phe-NH(2) (FMRFamide)-related peptides encoded by the genes flp-10 and flp-17. flp-10 is expressed in both neurons and non-neuronal cells. The major source of flp-17 peptides is a pair of presumptive sensory neurons, the BAG neurons. Genetic analysis of the egl-6 pathway revealed that the EGL-6 neuropeptide signaling pathway functions redundantly with acetylcholine to inhibit egg-laying. The retention of embryos in the uterus of the C. elegans hermaphrodite is therefore under the control of a presumptive sensory system and is inhibited by the convergence of signals from neuropeptides and the small-molecule neurotransmitter acetylcholine.     

FMRFamide-related peptide expression in the vestibular-afferent neurons

Vestibular-afferent neurons innervate hair cells from the sensory epithelia of vestibular end-organs and their action-potential discharge dynamics are driven by linear and angular accelerations of the head. The electrical activity of the vestibular-afferent neurons depends on their intrinsic properties and on the synaptic input from hair cells and from the terminals of the efferent system. Here we report that vestibular-afferent neurons of the rat are immunoreactive to RFamide-related peptides, and that the stronger signal comes from calyx-shaped neuron dendrites, with no signal detected in hair cells or supporting cells. The whole-cell voltage clamp recording of isolated afferent neurons showed that they express robust acid-sensing ionic currents (ASICs). Extracellular multiunit recordings of the vestibular nerve in a preparation in vitro of the rat inner ear showed that the perfusion of FMRFamide (a snail ortholog of this family of neuropeptides) exerts an excitatory effect on the afferent-neurons spike-discharge rate. Because the FMRFamide cannot activate the ASIC but reduces its desensitization generating a more robust current, its effect indicates that the ASIC are tonically active in the vestibular-afferent neurons and modulated by RFamide-like peptides.     

Processing of the precursor to the major core polypeptide of adenovirus type 5 removes a region near the amino terminus

The precursor to the major core polypeptide (pVII) of adenovirus type 5 has five methionine-containing tryptic peptides, while the mature core polypeptide (VII) after processing has only four. A technique utilizing the drug pactamycin has been used to determine the relative order (N terminal to C terminal) of the tryptic peptides within polypeptide pVII. Taking the order of these peptides into account and correlating it with the peptide missing from polypeptide VII, we are able to conclude that a region near the amino terminus of polypeptide pVII is removed during proteolytic processing.     

An immunocytochemistry study comparing the occurrence of neuroactive substances in the nervous system of cercariae and metacercariae of the eye flukeDiplostomum spathaceum

The nervous system of two larval stages (cercariae, metacercariae) of eye flukeDiplostomum spathaceum was investigated immunocytochemically by the application of antisera to the amino acid glutamate and to neuropeptides isolated from invertebrates (Mollusca) and from vertebrates to whole-mount preparations. In cercariae, positive immunoreactivity (IR) was observed with antisera raised against Catch-relaxing peptide (CARP), FMRFamide, -caudodorsal cell peptide (-CDCP), substance P, vasotocin, and vasopressin. In metacercariae, in addition to positive staining with these antisera, the ones raised against glutamate, APGWamide, caudodorsal cell hormone I (CDCH-I), and small cardiac peptide B (SCP_B) also gave positive IR in the nervous system. In the two larval stages the most extensive pattern of IR was observed with anti-FMRFamide and anti-CARP. In the nervous system of metacercariae the same immunoreactive neurosubstances appeared to be present as in that of cercariae. The increase in the variety of immunoreactive neurosubstances in the more complex nervous system of metacercariae is discussed in relation to parasite development and to host adaptation.     

Immunocytochemical demonstration of a homology in peptidergic neurosecretory cells in the suboesophageal ganglion of a beetle and a locust with antisera to bovine pancreatic polypeptide, FMRFamide, vasopressin and alpha-MSH

In the suboesophageal ganglion of the Colorado potato beetle and the migratory locust three types of peptidergic neurosecretory cells were identified immunocytochemically with antisera to bovine pancreatic polypeptide, FMRFamide, vasopressin and alpha-MSH. Their locations and immunocytochemical reactions are similar, which suggests that these peptidergic cells in both insect species are homologous and perhaps have similar functions.     

Pestivirus bovine viral diarrhea virus polypeptides: identification of new precursor proteins and alternative cleavage pathways.

The genome of pestivirus bovine viral diarrhea virus (BVDV) contains a single 11,964 nt long open reading frame (ORF) that is capable of encoding a 449 kDa putative polyprotein. Although previous studies have described many virus-coded polypeptides that are believed to arise by post- and/or co-translational proteolytic processing in infected cells, there are two separate regions in the ORF for which no polypeptide products could be identified. In the present study using site specific antisera, we identified two new large proteins of Mr 175 and 172 kDa which encompass two previously described smaller precursor proteins, p125 and p133, respectively. These two large precursor proteins together with two other previously described proteins p22 and gp118 (Mr approx. 84 kDa; unglycosylated) account for the predicted Mr of the putative 449 kDa polyprotein precursor. Furthermore, we discovered three additional polypeptides of Mr 168, 96 and 72 kDa encoded by the last third of the genome. These proteins may arise by an alternative cleavage pathway of the precursor protein p172. A modified and updated map of BVDV ORF incorporating the above findings is presented.