High identification rates of endogenous neuropeptides from mouse brain.

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 µL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.

Plasma and tissue levels of neuropeptide y in experimental septic shock: relation to hemodynamics, inflammation, oxidative stress, and hemofiltration.

Neuropeptide Y (NPY), a potent vasoconstrictor released from the sympathetic nerves, has been suggested to counterbalance sepsis-induced vasodilation. Thus, the changes in plasma and tissue NPY concentrations in relation to hemodynamic variables and inflammatory markers in a porcine model of moderate septic shock were investigated. Susceptibility of NPY to be removed by continuous hemofiltration in two settings has been also studied. Thirty-four domestic pigs were divided into five groups: (i) control group; (ii) control group with conventional hemofiltration; (iii) septic group; (iv) septic group with conventional hemofiltration; and (v) septic group with high-volume hemofiltration. Sepsis induced by fecal peritonitis continued for 22 h. Hemofiltration was applied for the last 10 h. Hemodynamic and inflammatory parameters (heart rate, mean arterial pressure, cardiac output, systemic vascular resistance, plasma concentrations of tumor necrosis factor-α, interleukin-6, and NPY) were measured before and at 12 and 22 h of peritonitis. NPY tissue levels were determined in the left ventricle and mesenteric and coronary arteries. Sepsis induced long-lasting increases in the systemic NPY levels without affecting its tissue concentrations. Continuous hemofiltration at any dose did not reduce sepsis-induced elevations in NPY plasma concentrations, nor did it affect the peptide tissue levels. The increases in NPY systemic levels were significantly correlated with changes in the systemic vascular resistance. The results support the hypothesis of NPY implication in the regulation of the vascular resistance under septic conditions and indicate that NPY clearance rate during hemofiltration does not exceed the capacity of perivascular nerves to release it.

Identification of neuropeptide Y-like conopeptides from the venom of Conus betulinus.

Neuropeptide Y (NPY) is a ubiquitous endocrine neuropeptide found in vertebrate and invertebrate. In our present work, two NPY-like exocrine conopeptides (designated as cono-NPYs) were first identified in the venom of cone snails. Both cono-NPYs showed sequence characteristics of invertebrate NPYs, suggesting that some exocrine venom peptides are probably evolved from the preexisting endocrine peptides during the evolution of cone snails.

Neuropeptide Y in the brain and retina of the adult teleost gilthead seabream (Sparus aurata L.).

The presence of neuropeptide Y (NPY) in the brain and retina of gilthead seabream (Sparus aurata L.) was investigated for the first time. For this investigation we employed an immunoperoxidase technique and the western immunoblot analysis using an antiserum raised against porcine NPY. The results showed that NPY-immunoreactivity was widely distributed in the brain of S. aurata. In particular, we have found NPY-immunoreactive (ir) neurons in the area ventralis telencephali pars centralis and pars lateralis, in the area dorsali telencephali pars centralis subdivision two and in nucleus intermedius thalami. An intense NPY-ir was detected in the telencephalon, in the optic tectum, in the thalamus, hypothalamus and in the vagal lobes. Scarce positive fibres were seen in the olfactory bulbs. NPY-ir amacrine cells were observed in the retina. The western immunoblot analysis revealed a protein band with a mobility corresponding to that of synthetic NPY. Our findings are, in general, in agreement with those obtained in other teleosts. The extensive distribution of NPY indicates for this peptide a key role in basic physiological actions, including visual and gustatory inputs processing.

Cloning, expression and growth promoting action of Red tilapia (Oreochromis sp.) neuropeptide Y.

Neuropeptide Y, a 36 amino acid peptide abundantly expressed in the brain, is the most potent orexigenic factor known to date in mammals. It has been shown to be one of the most conserved neuropeptides in vertebrate evolution. It seems that neuropeptide Y functions, in addition to sequence conservation, are also well conserved in fish. In the present study, we cloned and reported the cDNA sequence coding for tilapia 36 aminoacid neuropeptide Y. We express the tilapia neuropeptide Y gene in Escherichia coli driven by T7 promoter. The recombinant neuropeptide Y was purified up to 80% by affinity chromatography. We developed both, a food intake and a growth performance experiment to evaluate the effects of neuropeptide Y administration. Juvenile tilapia receiving recombinant neuropeptide Y (1 microg/g of body weight) by intraperitoneal injection increased food intake compared to controls (p < 0.05). Similarly, in the growth performance experiment, we observed an increase in body weight (p < 0.05) of tilapia fry receiving the same dose of the peptide. Neuropeptide Y treatment had no significant effect on hepatosomatic index and muscle moisture content. On the other hand, muscle protein content was increased in treated animals. These results demonstrate that administration of biologically active E. coli-derived neuropeptide Y resulted in a growth promoting action in fish.

Localization of Neuropeptide Y-like immunoreactive structures in the brain of the pejerrey, Odontesthes bonariensis (Teleostei, Atheriniformes).

The aim of this study was to determine the distribution of Neuropeptide-Y (NPY) immunoreactive neurons and fibres in the brain and pituitary of Odontesthes bonariensis by immunohistochemical methods. A wide distribution of immunoreactive NPY (ir-NPY) cells and fibres in the forebrain and midbrain was observed. A prominent ir-NPY nucleus was found in the ventral telencephalon and other ir-NPY cells groups were recognized at the dorso-medial telencephalon. The diencephalon showed ir-NPY cells in the Nucleus entopeduncularis, the Nucleus preopticus periventricularis and in the Nucleus lateralis tuberis. Ir-NPY fibres were conspicuous in the preoptic region and the hypothalamus. There were also numerous ir-NPY fibres at the epithalamic level running ventrally to the hypothalamus and the pituitary stalk. At the rhomboencephalic level, the ir-NPY neurons were observed in the Locus coeruleus. Double-labelled immunostaining showed a close association between ir-NPY fibres that reach the adenohypophysis and growth hormone (GH)- and gonadotropin (GtH)-expressing cells. Although our results exhibit some relevant differences when compared to other fish groups, they support the existence of a conserved NPY system in teleosts.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue.

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon.

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.

Capillary liquid chromatography of multiple peptides with on-line capillary electrophoresis immunoassay detection.

A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pM. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 microL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 microM and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.

Immunohistochemical mapping of enkephalins, NPY, CGRP, and GRP in the cat amygdala.

This immunohistochemical study shows a wide distribution of neuropeptides in the cat amygdala. Neuropeptide Y is present along the whole amygdaloid complex, and fibers and cell bodies containing neuropeptide Y are observed in all the nuclei studied. Leucine-enkephalin-, gastrin-releasing peptide/bombesin-, and calcitonin gene-related peptide-immunoreactive fibers and perikarya are observed only in discrete nuclei of the amygdaloid complex, whereas only fibers -but no cell bodies- containing methionine-enkephalin-Arg6-Gly7-Leu8 have been observed. No immunoreactivity has been found for gamma-melanocyte-stimulating hormone, dynorphin A (1-17), or galanin. These data are compared with those reported in the amygdala of other mammals.

Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis-mass spectrometry.

This paper describes the development of an analytical method for the separation and identification of neuropeptide Y (NPY) and two important NPY fragments by capillary electrophoresis (CE) and mass spectrometry (MS). A satisfactory separation and the highest sensitivity were obtained with formic acid at high concentrations (250 mM, pH 2.75). The addition of 25 or 50 mM triethylamine (TEA) improved the separation. When applying full scan CE-MS, the separated peptides could be detected and identified using the spectra of each peak. The use of TEA as an additive to the formic acid slightly decreased the sensitivity but was compensated by the improved efficiency. The best compromise for optimal separation and MS detection was found to be 50 mM formic acid to which 50 mM TEA was added. CE-MS could be used for identification of the decomposition products of NPY. Decomposition products with one amino acid difference, which could not be distinguished with CE-UV, could be distinguished with CE-MS.

Neuropeptide Y: a novel angiogenic factor from the sympathetic nerves and endothelium.

Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the "NPY-converting enzyme" dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr1-Pro2 from NPY to form an angiogenic Y2 agonist, NPY3-36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.

Analytical extraction of regulatory peptides from rat lung tissue.

We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and beta-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and beta-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.

Effects of covalent dimerization on the structure and function of the carboxy-terminal fragment of neuropeptide Y.

To determine whether or not the dimeric structure of neuropeptide Y (NPY) that is found in solution is necessary for its function, we investigated the effects of covalent dimerization on the structure and function of NPY using the carboxy-terminal fragment, NPY(12-36), in which residues 12 and 31 (located at both ends of alpha-helical region) were replaced by Cys residues. Among the three species (the parallel dimer, the anti-parallel dimer, and the intramolecularly cross-linked monomer) obtained by oxidation of the fragment, the anti-parallel dimer was predominant. NMR analysis showed that both parallel and anti-parallel dimers had alpha-helices similar to that of intact NPY, suggesting that covalent dimerization might have little effect on the helical structure. A binding assay with Y2 receptors on porcine hippocampal membranes revealed that the IC50 value of the anti-parallel dimer was almost the same as that of NPY (13-36), which is known as a Y2-specific ligand. By contrast, the binding by the parallel dimer was weaker by more than one order of magnitude. Our results suggest that the formation of dimers of NPY is not essential for binding to the receptor.

Neuropeptide Y 3-36 is an endogenous ligand selective for Y2 receptors.

Neuropeptide Y (NPY 1-36) binds to Y1 and Y2 receptors with similar affinity. No endogenous molecular form of NPY with selectivity for Y1 or Y2 receptors has been described so far. We report the presence of an endogenous fragment of NPY in porcine brain, NPY 3-36, which lacks the amino-terminal dipeptide Tyr-Pro of NPY 1-36. NPY 3-36 accounts for 35% of NPY-like immunoreactivity in porcine brain. We have compared binding of NPY 3-36 and NPY 1-36 in model systems of Y1-like (SK-N-MC cells) and Y2-like receptors (CHP234 cells). NPY 3-36 and NPY 1-36 had similarly high affinity for Y2-like receptors on CHP234 cells, but NPY 3-36 had a 1000-fold lower affinity than NPY 1-36 for Y1-like receptors on SK-N-MC cells. Thus amino-terminal cleavage of NPY 1-36 generating NPY 3-36 converts an unselective Y1/Y2 receptor ligand into a highly Y2 selective ligand. This may be a means of fine tuning NPY biological actions.

Synergistic action of neuropeptide Y and adrenaline in the eel atrium.

To investigate the influence of neuropeptide Y (NPY) on heart function in a relatively simple model system, the effects of eel NPY (eNPY) on the eel atrium were examined. Eel NPY enhanced the contractile force of the isolated atrium in a dose-dependent manner, without altering the rate of contraction. Although adrenaline also exerts a positive inotropic effect, the effect of eNPY was not blocked by the beta 1-adrenoceptor antagonist betaxolol, indicating that eNPY does not act via adrenaline release. When eNPY and adrenaline were applied simultaneously, their effects were additive at lower concentrations but not at higher concentrations. The plateau reached at high concentrations suggests that these two regulators act through a common signal transduction process. One candidate for this is an elevation of the concentration of intracellular free Ca2+ ([Ca2+]i), since treatment with eNPY or adrenaline enhanced [Ca2+]i, as assessed by fluorescence of Calcium Green-1. The increase in [Ca2+]i after eNPY and adrenaline treatment is presumably due to Ca2+ influx from the external medium, since the effect was greatly reduced in Ca(2+)-free Ringer's solution and after treatment with verapamil, a Ca2+ channel blocker. Although both eNPY and adrenaline enhanced the atrial contractile force, the time courses were different, with the effect of eNPY being gradual and long-lasting, and that of adrenaline being immediate and transient. It is plausible, therefore, that eNPY and catecholamine(s) act synergistically to cause a long-lasting enhancement of contraction force if the two regulators arrive at the atrium simultaneously. The present study also demonstrates that the eel heart contains eNPY.

Effects of eel neuropeptide Y on ion transport across the seawater eel intestine.

A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (Isc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the Isc were concentration-dependent with a threshold concentration of 3 x 10(-9) M and a maximal effect at 3 x 10(-7) M. Similar concentration-response curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an alpha 2-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3 x 10(-8) M for eNPY, 3 x 10(-8) M for AD), but not at high dosage (10(-6) M eNPY, 10(-7) M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na(+)-K(+)-Cl- cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na(+)-K(+)-Cl- cotransporter.