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PubMed2000

Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor.

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

Amino Acid Sequence↗
PubMed2002

Peptidomics of the larval Drosophila melanogaster central nervous system.

Neuropeptides regulate most, if not all, biological processes in the animal kingdom, but only seven have been isolated and sequenced from Drosophila melanogaster. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomics approach aims at the simultaneous identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their posttranslational modifications. Using nanoscale liquid chromatography combined with tandem mass spectrometry and data base mining, we analyzed the peptidome of the larval Drosophila central nervous system at the amino acid sequence level. We were able to provide biochemical evidence for the presence of 28 neuropeptides using an extract of only 50 larval Drosophila central nervous systems. Eighteen of these peptides are encoded in previously cloned or annotated precursor genes, although not all of them were predicted correctly. Eleven of these peptides were never purified before. Eight other peptides are entirely novel and are encoded in five different, not yet annotated genes. This neuropeptide expression profiling study also opens perspectives for other eukaryotic model systems, for which genome projects are completed or in progress.

Amino Acid Sequence↗
PubMed2003

Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions.

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Animals↗
PubMed2004

Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons.

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.

Acid Sensing Ion Channels↗
PubMed2006

Changes in FaRP-like peptide levels during development of eggs from the plant-parasitic cyst nematode, Heterodera glycines.

The plant-parasitic cyst nematode Heterodera glycines requires a host plant to complete its life cycle, which involves hatching of infective juveniles that parasitize through root entry. A laboratory population of H. glycines grown on soybean, Glycine max, undergoes a sharp increase in maturity between 5 and 6 weeks in culture, as measured by the proportion of eggs containing well developed pre-hatch juveniles (late development eggs) versus eggs without visible juveniles (early development eggs). The median percent of eggs classified as late development, representing all samples taken from 4 to 7 weeks in culture, was 61%. For all samples taken up to 5 weeks, 80% scored below the median. In samples taken after 5 weeks, 15% scored below the median. This shift in population maturity was accompanied by a significant increase (P < 0.01) in the number of hatched juveniles present in each sample. There was also a significant increase (P < 0.02) in amount of FaRP-like peptide detected by specific ELISA. Total FaRP levels increased from 0.18 +/- 0.07 fMol FLRFamide equivalents per ng protein in early development eggs to 0.40 +/- 0.17 in late development eggs. The level remained high in hatched juveniles. HPLC/ELISA detected as many as nine potential FaRPs in H. glycines, two of which were specifically increased (P < 0.005) in hatched juveniles. The association of FaRPs with maturing eggs and the possible involvement of these neuropeptides with juvenile hatching and motility are discussed.

Animals↗
PubMed1995

Conformational preferences of the calliFMRFamides and their free-acid analogues.

A molecular dynamics study was undertaken to determine the conformational basis for the differing activities of the insect neuropeptide hormones calliFMRFamide 3 (SPSQDFMRF-NH2). calliFMRFamide 5 (APGQDFMRF-NH2) and their corresponding free-acid analogues (SPSQDFM-RF-OH and APGQDFMRF-OH) in two insect bioassays. A simulated annealing protocol was used to determine the range of conformers available to the linear peptides. Analysis of the conformers obtained indicated that all the peptides exhibited distinct secondary structure preferences. These, when correlated with their biological activities, enabled the formulation of putative conformation-activity relationships for the peptides.

Amino Acid Sequence↗
PubMed1986

Localization of Aplysia neurosecretory peptides to multiple populations of dense core vesicles.

Many neurons in the mollusc Aplysia are identifiable and provide a useful model system for investigating the cellular mechanisms used by the neuroendocrine system to mediate simple behaviors. In this study we determined the subcellular localization of eight Aplysia neuropeptides using immunogold labeling techniques, and analyzed the size distribution of dense core and granular vesicles in peptidergic neurons. Recent observations demonstrate that many neurons use multiple chemical messengers. Thus, an understanding of the functional significance of cotransmitters requires an analysis of their relative subcellular distributions. The peptides are expressed in a subset of neurons, or the exocrine atrial gland, and are primarily localized to dense core vesicles. Multiple regions of precursors which are cleaved into several components are co-localized. Each neuron has a distinct size distribution of peptide-containing dense core vesicles ranging in size from 65 to 600 nm. The atrial gland contains very large (up to 2 micron) peptide-containing granules. Single neurons have multiple populations of granules whose quantal sizes agree with predictions based on physical constraints. Some cells contain very large peptide-containing granules which are found in the cell soma and not in processes. Thus, the genetic determination of neuronal cell type includes not only transmitter choices but also multiple modes of packaging the intercellular messengers.

Animals↗
PubMed1997

Phe-Met-Arg-Phe-amide activates a novel voltage-dependent K+ current through a lipoxygenase pathway in molluscan neurones.

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED50 = 23 nM) activated a K+ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K+]o = 1.7 mM), only outward current responses occurred. In high K+ salines ([K+]o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K+. In both salines, no responses were measured below -120 mV. Between -120 mV and the K+ reversal potential, currents were inward with maximal amplitudes at approximately -60 mV. Thus, U-shaped current-voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K+ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per approximately 14 mV at all [K+]o. Since this result was also obtained in nearly symmetrical K+ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K+ concentrations. Onset kinetics of the response were slow (rise time approximately 650 ms at -40 mV). However, when FMRFa was applied while holding the cell at -120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to -40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at -120 mV, the time constant of activation during the subsequent test pulse decreased from approximately 36 ms at -60 mV to approximately 13 ms at -30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from -120 to -50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba2+, and, partially, Cd2+ and Cs+. The response to FMRFa was affected by intracellular GTPgammaS. The response was inhibited by blockers of phospholipase A2 and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K+ current distinguish it from other receptor-driven K+ currents such as the S-current- and G-protein-dependent inward rectifiers.

Animals↗
PubMed2001

Exploration of the pore structure of a peptide-gated Na+ channel.

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Amino Acid Sequence↗
PubMed1991

The olfactoretinalis system = terminal nerve?

Immunohistochemistry revealed a distinct terminal nerve (nT) from the olfactoretinalis system (ORS) in several gymnotid species. The ganglion (NOR) of the ORS is a cluster of FMRF-amide immunoreactive neurones located on the olfactory nerve and bulb. The NOR projects to the olfactory bulb and optic nerve but not to the olfactory epithelium. In contrast, the nT is a small substance P or GnRH immunoreactive fibre bundle which originates from a ganglion located rostral to the olfactory epithelium, courses caudally with the olfactory nerve and terminates as glomeruli in the olfactory bulb. Peripherally the ganglion projects to the epithelium of the anterior nostril. Thus, the terminal nerve appears to be a cranial nerve, clearly distinct from the ORS which consequently should no longer be considered as the terminal nerve.

Animals↗
PubMed1988

Radioimmunoassay for rat pancreatic alpha-amylase and the effect of Phe-Met-Arg-Phe-amide on amylase secretion in the isolated perfused rat pancreas.

In this study a radioimmunoassay was developed to measure secreted amylase from the isolated perfused rat pancreas. Using Sephadex G-75 gel chromatography, rat pancreatic amylase was purified to a single migrating protein band as determined by SDS polyacrylamide gel electrophoresis. Specificity of a rat pancreatic amylase antiserum, raised in rabbits, was determined using immunodiffusion, immunoelectrophoresis, and immunoblotting techniques. Secreted amylase concentrations, obtained using the radioimmunoassay, were not significantly different than those measured with the amylase enzyme assay. The rat pancreatic amylase radioimmunoassay was used to measure the amylase secretion in the isolated perfused rat pancreas. Phe-Met-Arg-Phe-amide (FMRF-NH2) immunoreactivity has been shown to be co-localized with pancreatic polypeptide in the rat pancreatic islet, and evidence suggests that islet peptides modulate amylase secretion from the exocrine pancreas. In the present study, FMRF-NH2 significantly (p less than 0.05) suppressed cholecystokinin (CCK)-stimulated amylase secretion by 55%. The average pancreatic amylase secretion in response to CCK was 10.89 +/- 2.0 micrograms/ml/min (n = 6); with the addition of FMRF-NH2, CCK-stimulated amylase secretion was reduced to 4.79 +/- 1.6 micrograms/ml/min (n = 6). These results are consistent with the insuloacinar hypothesis in that an FMRF-NH2-like substance in the islet may act to modulate the exocrine pancreas.

Animals↗
PubMed1985

Characterization of FMRF amide-like immunoreactivity in rat spinal cord by region-specific antibodies in radioimmunoassay and HPLC.

Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe-Met-Arg-Phe-NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N-terminally extended analogue, Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C-terminus; both required the C-terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C-terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed-phase HPLC using Techsil C18 and Spherisorb-phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C-terminal tripeptide of FMRF amide and are probably extended at the N-terminus by sequences immunochemically distinct from other known peptides.

Animals↗
PubMed2005

LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis.

We report the characterization of a cDNA encoding a novel -RFamide neuropeptide precursor that is up-regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C-terminal sequence -LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+-currents, and inhibited electrical activity. We conclude that up-regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.

Animals↗