Peptides (v.29, #12)

Announcement (VII).

Structure–activity studies of AtPep1, a plant peptide signal involved in the innate immune response by Gregory Pearce; Yube Yamaguchi; Gerhard Munske; Clarence A. Ryan (2083-2089).
AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure–activity relationship of AtPep1 with its receptor, a 170 kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15 min with a half-maximal response of 0.25 nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9–23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9–23), resulted in a decrease in activity ( 1 2 max ∼ 100 nM). AtPep1(9–23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A15]AtPep1(9–23) ( 1 2 max ∼ 10 nM), and a glycine, [A17]AtPep1(9–23) ( 1 2 max ∼ 1000 nM). Neither [A17]AtPep1(9–23) nor the C-terminal truncated AtPep1, AtPep1(9–22), were able to compete with AtPep1(9–23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d-Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l- and d-proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.
Keywords: AtPep; Plant defense; Peptide signaling; Innate immunity;

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds by Patrícia D. Games; Izabela S. dos Santos; Érica O. Mello; Mariângela S.S. Diz; André O. Carvalho; Gonçalo A. de Souza-Filho; Maura Da Cunha; Ilka M. Vasconcelos; Beatriz dos S. Ferreira; Valdirene M. Gomes (2090-2100).
The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301–9], with some modifications. A DEAE-Sepharose, equilibrated with 20 mM Tris–HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS–Tricine gel electrophoresis with a molecular mass of approximately 6 kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314 bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.
Keywords: Antimicrobial peptides; Plant defensin; Pathogenic yeasts; Phytopathogenic fungi; Molecular cloning; Phylogenetic analysis;

Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta by Linda S.M. Ooi; Li Tian; Miaoxian Su; Wing-Shan Ho; Samuel S.M. Sun; Hau-Yin Chung; Henry N.C. Wong; Vincent E.C. Ooi (2101-2109).
A fetuin-binding peptide with a molecular mass of about 9 kDa (designated NTP) was isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L., by gel filtration and high-performance liquid chromatography, after removing the mannose-binding proteins by mannose–agarose column. Molecular cloning revealed that NTP contained an open reading frame of 354 bp encoding a polypeptide of 118 amino acids which included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays, NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60).
Keywords: Narcissus tazetta; Non-specific lipid transfer proteins; Respiratory syncytial virus; H1N1 virus; HL-60 cells;

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds by Seiya Yokoyama; Kouji Kato; Atsuko Koba; Yuji Minami; Keiichi Watanabe; Fumio Yagi (2110-2117).
Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.
Keywords: Cycas revoluta; Antimicrobial activity; Chitin binding domain; nsLTP; Plant defensin;

Parotid secretory protein (PSP) (SPLUNC2), a potential host-defense protein related to bactericidal/permeability-increasing protein (BPI), was used as a template to design antibacterial peptides. Based on the structure of BPI, new PSP peptides were designed and tested for antibacterial activity. The peptides did not exhibit significant bactericidal activity or inhibit growth but the peptide GL-13 induced bacterial matting, suggesting passive agglutination of bacteria. GL-13 was shown to agglutinate the Gram negative bacteria Pseudomonas aeruginosa and Aggregatibacter (Actinobacillus) actinomycetemcomitans, Gram positive Streptococcus gordonii and uncoated sheep erythrocytes. Bacterial agglutination was time and dose-dependent and involved hydrophobic interactions. Variant forms of GL-13 revealed that agglutination also depended on the number of amine groups on the peptide. GL-13 inhibited the adhesion of bacteria to plastic surfaces and the peptide prevented the spread of P. aeruginosa infection in a lettuce leaf model, suggesting that GL-13 is active in vivo. Moreover, GL-13-induced agglutination enhanced the phagocytosis of P. aeruginosa by RAW 264.7 macrophage cells. These results suggest that GL-13 represents a class of antimicrobial peptides, which do not directly kill bacteria but instead reduce bacterial adhesion and promote agglutination, leading to increased clearance by host phagocytic cells. Such peptides may cause less bacterial resistance than traditional antibiotic peptides.
Keywords: Agglutination; Antimicrobial peptide; GL-13; PSP; SPLUNC2; C20orf70; PLUNC;

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH2) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH2) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH2, and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH2, and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.
Keywords: Cholecystokinin; Drosulfakinin; Feeding; Odorants; Olfactory;

A peptide of the phylloseptin family from the skin of the frog Hylomantis lemur (Phyllomedusinae) with potent in vitro and in vivo insulin-releasing activity by Yasser H.A. Abdel-Wahab; Gavin J. Power; Peter R. Flatt; Douglas C. Woodhams; Louise A. Rollins-Smith; J. Michael Conlon (2136-2143).
A peptide with the ability to release insulin from the rat BRIN-BD11 clonal β cell line was isolated from norepinephrine-stimulated skin secretions of the Lemur leaf frog Hylomantis lemur Boulenger,1882. Determination of the primary structure (FLSLIPHVISALSSL.NH2) demonstrated that the peptide belongs to the phylloseptin family whose members have previously been identified in other Phyllomedusinae species. A synthetic replicate of the peptide, termed phylloseptin-L2, produced a significant stimulation of insulin release (134% of basal rate, P  < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (301% of basal rate, P  < 0.001) at a concentration of 3 μM. Phylloseptin-L2 did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. The stimulatory action was maintained in the absence of extracellular Ca2+ and in the presence of verapamil (50 μM) and diazoxide (300 μM) suggesting that mechanism of action of the peptide did not primarily involve influx of Ca2+ or closure of ATP-sensitive K+ channels. Administration of phylloseptin-L2 (50 nmol/kg body weight) into mice significantly (P  < 0.05) increased total release of insulin and improved glucose tolerance during the 60 min period following an intraperitoneal injection of glucose (18 mmol/kg body weight). It is concluded that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.
Keywords: Frog skin; Insulin-releasing peptide; Phyllomedusinae; Phylloseptin;

Bolus administration of obestatin does not change glucose and insulin levels neither in the systemic nor in the portal circulation of the rat by Rosalie M. Kiewiet; Carlotta Gauna; Maarten O. van Aken; Bedette van de Zande; Aart Jan van der Lely (2144-2149).
Obestatin is a second peptide derived from the preproghrelin polypeptide. It was originally thought to have anorexigenic effects, thereby functioning as an antagonist of ghrelin. However, this has been a subject of debate ever since. Since acylated ghrelin strongly induces insulin resistance, it could be hypothesized that obestatin plays a role in glucose homeostasis as well. In the present study we evaluated the effect of obestatin on glucose and insulin metabolism in the systemic and portal circulation. Obestatin 200 nmol/kg was administered systemically as a single intravenous bolus injection to fasted pentobarbital anesthetized adult male Wistar rats. Up to 50 min after administration, blood samples were taken to measure glucose and insulin concentrations, both in the portal and in the systemic circulation. The effect of obestatin was evaluated in fasted and in glucose-stimulated conditions (IVGTT) and compared to control groups treated with saline or IVGTT, respectively. Intravenous administration of obestatin did not have any effect on glucose and insulin concentrations, neither systemic nor portal, when compared to the control groups. Only the glucose peak 1 min after administration of IVGTT was slightly higher in the obestatin treated rats: 605.8 ± 106.3% vs. 522.2 ± 47.1% in the portal circulation, respectively (NS), and 800.7 ± 78.7% vs. 549.6 ± 37.0% in the systemic circulation, respectively (P  < 0.02), but it can be debated whether this has any clinical relevance. In the present study, we demonstrated that intravenously administered obestatin does not influence glucose and insulin concentrations, neither in the portal nor in the systemic circulation.
Keywords: Obestatin; Ghrelin; Wistar rat; Glucose; Insulin; IVGTT;

Ghrelin and obestatin: Different role in fetal lung development? by Susana Nunes; Cristina Nogueira-Silva; Emanuel Dias; Rute S. Moura; Jorge Correia-Pinto (2150-2158).
Ghrelin and obestatin are two proteins that originate from post-translational processing of the preproghrelin peptide. Various authors claim an opposed role of ghrelin and obestatin in several systems. Preproghrelin mRNA is significantly expressed in airway epithelium throughout lung development, predominantly during the earliest stages. The aim of this study was to evaluate the role of ghrelin and obestatin in fetal lung development in vitro. Immunohistochemistry studies were performed at different gestational ages in order to clarify the expression pattern of ghrelin, GHS-R1a, obestatin and GPR39 during fetal lung development. Fetal rat lung explants were harvested at 13.5 days post-conception (dpc) and cultured during 4 days with increasing doses of total ghrelin, acylated ghrelin, desacyl-ghrelin, ghrelin antagonist (D-Lys(3)-GHRP-6) or obestatin. Immunohistochemistry studies demonstrated that ghrelin, GHS-R1a, obestatin and GPR39 proteins were expressed in primitive rat lung epithelium throughout all studied gestational ages. Total and acylated ghrelin supplementation significantly increased the total number of peripheral airway buds, whereas desacyl-ghrelin induced no effect. Moreover, GHS-R1a antagonist significantly decreased lung branching. Finally, obestatin supplementation induced no significant effect in the measured parameters. The present study showed that ghrelin has a positive effect in fetal lung development through its GHS-R1a receptor, whereas obestatin has no effect on lung branching.
Keywords: Ghrelin; Obestatin; GHS-R1a; GPR39; Fetal lung; Development;

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats by Tobias Inhoff; Hubert Mönnikes; Steffen Noetzel; Andreas Stengel; Miriam Goebel; Q. Thai Dinh; Andrea Riedl; Norbert Bannert; Anna-Sophia Wisser; Bertram Wiedenmann; Burghard F. Klapp; Yvette Taché; Peter Kobelt (2159-2168).
Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 μg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 μg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12 h. Ghrelin and desacyl ghrelin (64 μg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.
Keywords: Rat; Desacyl ghrelin; Ghrelin; Food intake; c-Fos; ARC;

Corticotropin-releasing factor (CRF) is involved in the acute anorexic effect of α-melanocyte-stimulating hormone: A study using CRF-deficient mice by Shoko Kawashima; Satoru Sakihara; Kazunori Kageyama; Takeshi Nigawara; Toshihiro Suda (2169-2174).
Alpha-melanocyte-stimulating hormone (α-MSH) and its receptors are critical and indispensable for maintaining appropriate feeding behavior and energy homeostasis in both mice and humans. Corticotropin-releasing factor (CRF) is a candidate for mediating the anorexic effect of α-MSH. In the present study, we examined whether CRF and its receptors are involved in the anorexic effect of α-MSH, using CRF-deficient (CRFKO) mice and a CRF receptor antagonist. Intracerebroventricular administration of NDP-MSH, a synthetic α-MSH analogue, suppressed food intake in wild-type (WT) mice. This effect was abolished by pretreatment with a non-selective CRF receptor antagonist, astressin, suggesting that the effect of α-MSH-induced anorexia was mediated by a CRF receptor. In CRFKO mice, administration with NDP-MSH did not affect food intake at an early phase (0–4 h). In addition, CRF mRNA levels in the hypothalamus were significantly increased in NDP-MSH-treated mice. Therefore, our findings, using CRFKO, strongly support evidence that CRF is involved in the acute anorexic effect of α-MSH. On the other hand, NDP-MSH administered to CRFKO mice led to suppressed food intake at the late phase (4–12 h), similar to the effect in WT mice. Further, NDP-MSH similarly reduced food intake during the late phase in all types of mice, including WT, CRFKO, and CRFKO with corticosterone replacement. The results would suggest that α-MSH-induced suppression of food intake at late phase was independent of glucocorticoids and CRF.
Keywords: Melanocyte-stimulating hormone; Corticotropin-releasing factor; Feeding; Anorexia;

Enterostatin reduces serum cholesterol levels by way of a CCK1 receptor-dependent mechanism by Yasuyuki Takenaka; Tomoko Shimano; Takaaki Mori; I-ching Hou; Kousaku Ohinata; Masaaki Yoshikawa (2175-2178).
Enterostatin (APGPR), an anorectic pentapeptide derived from the amino terminus of procolipase, significantly reduced serum cholesterol levels after oral administration at a dose of 100 mg/kg for 3 days in mice fed a high-cholesterol-cholic acid diet. The hypocholesterolemic effect of APGPR was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK1) receptor, even though APGPR does not have any affinity for CCK1 receptors. Similarly, the hypocholesterolemic activity of VPDPR, an APGPR analogue, was blocked by lorglumide. These results suggest that the hypocholesterolemic effects of APGPR and VPDPR are mediated by a CCK1 receptor-dependent mechanism.
Keywords: Enterostatin; APGPR; VPDPR; Hypocholesterolemic effect; CCK1 receptor;

The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age by Mirjana Dimitrijević; Stanislava Stanojević; Katarina Mitić; Nataša Kuštrimović; Vesna Vujić; Tatjana Miletić; Vesna Kovačević-Jovanović (2179-2187).
Neuropeptide Y (NPY)-induced modulation of the immune and inflammatory responses is regulated by tissue-specific expression of different receptor subtypes (Y1–Y6) and the activity of the enzyme dipeptidyl peptidase 4 (DP4, CD26) which terminates the action of NPY on Y1 receptor subtype. The present study investigated the age-dependent effect of NPY on inflammatory paw edema and macrophage nitric oxide production in Dark Agouti rats exhibiting a high-plasma DP4 activity, as acknowledged earlier. The results showed that NPY suppressed paw edema in adult and aged, but not in young rats. Furthermore, plasma DP4 activity decreased, while macrophage DP4 activity, as well as macrophage CD26 expression increased with aging. The use of NPY-related peptides and Y receptor-specific antagonists revealed that anti-inflammatory effect of NPY is mediated via Y1 and Y5 receptors. NPY-induced suppression of paw edema in young rats following inhibition of DP4 additionally emphasized the role for Y1 receptor in the anti-inflammatory action of NPY. In contrast to the in vivo situation, NPY stimulated macrophage nitric oxide production in vitro only in young rats, and this effect was mediated via Y1 and Y2 receptors. It can be concluded that age-dependant modulation of inflammatory reactions by NPY is determined by plasma, but not macrophage DP4 activity at different ages.
Keywords: Dark Agouti rats; Dipeptidyl peptidase 4 (DP4); Inflammation; Macrophages; Neuropeptide Y (NPY); Nitric oxide;

Peptidomic analysis of blood plasma after in vivo treatment with protease inhibitors—A proof of concept study by Harald Tammen; Rüdiger Hess; Horst Rose; Wolfgang Wienen; Marco Jost (2188-2195).
Native peptides can be regarded as surrogate markers for protease activity in biological samples. Analysis of peptides by peptidomics allows to monitor protease activity in vivo and to describe the influence of protease inhibition. To elucidate the potential of peptides as markers for in vivo protease inhibition we analyzed plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX™ or the dipeptidylpeptidase IV inhibitor AB192. Signals correlating with the treatment were subsequently identified and assessed with respect to protease-dependent consensus cleavage motifs and occurrence of downstream targets. It could be shown that regulated peptides were either substrates, products or downstream targets of the inhibited protease. The results from the present study demonstrate that the in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor related effects in vivo and that this method may pave the way to develop predictive biomarkers.
Keywords: Peptidomics; DPPIV; Dipetidylpeptidase IV; FONDAPARINUX; Protease inhibition; Factor Xa;

Pharmacokinetics of proline-rich tripeptides in the pig by Pieter C. van der Pijl; Arie K. Kies; Gabriella A.M. Ten Have; Guus S.M.J.E. Duchateau; Nicolaas E.P. Deutz (2196-2202).
Tripeptides may possess bioactive properties. For instance, blood pressure lowering is attributed to the proline-rich tripeptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP), and Val-Pro-Pro (VPP). However, little is known about their absorption, distribution, and elimination characteristics. The aim of this study was to characterize the pharmacokinetic behavior of IPP, LPP, and VPP in a conscious pig model. Synthetic IPP, LPP, and VPP were administered intravenously or intragastrically (4.0 mg kg−1 BW in saline) to 10 piglets (approximately 25 kg body weight) in the postabsorptive state. After intravenous dosing, the elimination half-life for IPP was significantly higher (P  < 0.001) than for LPP and VPP (2.5 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 min, respectively). After intragastric dosing, however, the elimination half-lives were not significantly different between the peptides (9 ± 1, 15 ± 4, and 12 ± 6 min, respectively). Maximum plasma concentrations were about 10 nmol l−1 for the three tripeptides. The fraction dose absorbed was 0.077 ± 0.010, 0.059 ± 0.009, and 0.073 ± 0.015%, for IPP, LPP, and VPP, respectively. Proline-rich tripeptides reach the blood circulation intact, with an absolute bioavailability of about 0.1% when administered via a saline solution. Because half-lives of absorption and elimination were maximally about 5 and 15 min, respectively, this suggests that under these conditions a bioactive effect of these tripeptides would be rather acute.
Keywords: Absolute bioavailability; Bioactive peptides; Ile-Pro-Pro; Leu-Pro-Pro; Val-Pro-Pro; Interorgan pig model;

Biosynthesis and secretion of salusin-α from human cells by Kengo Sato; Takatoshi Koyama; Masayoshi Shichiri (2203-2207).
Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-α is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-α levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-α peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-α in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-α peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-α-like immunoreactivity to be present in HeLa cells which released salusin-α-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-α. These results present the first evidence that salusin-α is biosynthesized and released from human-derived cells.
Keywords: Salusin-α; Radioimmunoassay; Reverse-phase high performance liquid chromatography; Processing; Immunohistochemistry; Secretion;

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart by Silvia Del Ry; Manuela Cabiati; Vincenzo Lionetti; Michele Emdin; Fabio A. Recchia; Daniela Giannessi (2208-2215).
C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n  = 5) and with pacing-induced HF (n  = 5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9 ± 10.4 pg/ml vs.16.7 ± 1.1, p  = 0.013, mean ± S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7 ± 1.9 pg/mg protein; LA: 8.7 ± 3.8; RV: 1.07 ± 0.33; LV: 0.93 ± 0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8 ± 9.9 pg/mg protein vs. 0.9 ± 0.17, p  = 0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.
Keywords: C-type natriuretic peptide; Natriuretic peptide; Natriuretic peptide receptors; Chronic heart failure;

Stimulation of ANP secretion by 2-Cl-IB-MECA through A3 receptor and CaMKII by Kuichang Yuan; Guang Yi Bai; Woo Hyun Park; Sung Zoo Kim; Suhn Hee Kim (2216-2224).
Adenosine is a potent mediator of myocardial protection against hypertrophy via A1 or A3 receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A3 receptor on ANP release. We studied the effects of the A3 receptor on atrial functions and its modification in hypertrophied atria. A selective A3 receptor agonist, 2-chloro-N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A3 receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30 μM 2-CI-IB-MECA were not affected. The 100 μM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA), A3 receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA ≫ IB-MECA = HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca2+ channels, sarcoplasmic reticulum Ca2+-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A3 receptor activation and positive inotropy by intracellular Ca2+ regulation via the ryanodine receptor and CaMKII.
Keywords: A3 receptor; Adenosine; Purinoceptor; Atrial natriuretic peptide; Atrial contractility; cAMP; Signal transduction;

A possible association between aldosterone response to vasopressin and circadian change of aldosterone in the patients with aldosterone-producing adenoma by Sawako Suzuki; Daigaku Uchida; Hisashi Koide; Keiko Suyama; Takahisa Shibata; Tomohiko Yoshida; Tomoaki Tanaka; Yoshihiko Noguchi; Yasushi Saito; Ichiro Tatsuno (2225-2231).
Vasopressin was reported to stimulate secretion of both cortisol and aldosterone through eutopic V1a receptors in adrenal gland. Recently, adrenal hyper-responsiveness of plasma cortisol to vasopressin with eutopic overexpession of V1a receptors has been reported in Cushing’s syndrome, such as a majority of cases of ACTH-independent macronodular adrenal hyperplasia and some cases of Cushing’s adenomas. There were a few reports regarding the aldosterone response to vasopressin in aldosterone-producing adenoma. The aim of our study was to investigate the aldosterone response to vasopressin and its pathophysiological roles in the patients with aldosterone-producing adenoma. Vasopressin-loading test was performed in 10 patients with aldosterone-producing adenoma, and in 16 patients with non-functioning adrenal tumors. The roles of the aldosterone response to vasopressin were analyzed in terms of hormonal secretion and the expression of V1a receptor mRNA on the operated adrenal gland in aldosterone-producing adenoma. We found that (1) a varying aldosterone response to vasopressin was observed, (2) absolute response of plasma aldosterone in aldosterone-producing adenoma was significantly higher than that in non-functioning tumor, (3) aldosterone response rate to vasopressin was significantly and negatively correlated with the decline rate (%) in plasma aldosterone from morning to evening in aldosterone-producing adenoma, (4) V1a receptor mRNA was expressed at various values in aldosterone-producing adenoma, and (5) surgical removal of aldosterone-producing adenoma eliminated the aldosterone response to vasopressin observed in patients with aldosterone-producing adenoma. These findings indicated that vasopressin might be involved in the coordination of aldosterone secretion through eutopic expression of V1a receptor in aldosterone-producing adenoma.
Keywords: Vasopressin; Aldosterone; Aldosterone-producing adenoma; V1a receptor; Circadian change;

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation by Valeria Marsili; Giulio Lupidi; Giuliano Berellini; Isabella Calzuola; Stefano Perni; Gabriele Cruciani; Gian Luigi Gianfranceschi (2232-2242).
DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide–N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.
Keywords: Chromatin peptides; N-CAM; Fluorescence quenching; Peptide–protein interaction; Peptide–peptide interaction; Neuronal differentiation;

Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo by William P. Dunworth; Kimberly L. Fritz-Six; Kathleen M. Caron (2243-2249).
The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM −/− endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins.
Keywords: Adrenomedullin; VEGFA; Permeability; Lymphatic; Endothelial; Lymphography;

Regulation of glial inflammatory mediators synthesis: Possible role of endothelins by Talia Filipovich; Sigal Fleisher-Berkovich (2250-2256).
Endothelins are well known as modulators of inflammation in the periphery, but little is known about their possible role in brain inflammation. Stimulation of astrocyte prostaglandin, an inflammatory mediator, synthesis was shown so far only by endothelin 3 (ET-3). By contrast, several studies showed no change or slight decrease of basal nitric oxide synthesis after treatment of astrocytes with endothelin 1 (ET-1) and ET-3. However, a significant increase in astrocytic and microglial nitric oxide synthase (NOS) was observed after exposure to ET-1 and ET-3 in a model of forebrain ischaemia. Here we demonstrate that all three endothelins (ET-1, ET-2, ET-3) significantly enhanced the synthesis of prostaglandin E2 and nitric oxide in glial cells. Each of the selective antagonists for ETA and ETB receptors (BQ123 and BQ788 respectively), significantly inhibited endothelins-induced production of both nitric oxide and prostaglandin E2. These results suggest a regulatory mechanism of endothelins, interacting with both endothelin receptors, on glial inflammation. Therefore, inhibition of endothelin receptors may have a therapeutic potential in pathological conditions of the brain, when an uncontrolled inflammatory response is involved.
Keywords: Endothelins; Prostaglandin E2; Nitric oxide; Glial cells; Brain inflammation;

Nocistatin and nociceptin given centrally induce opioid-mediated gastric mucosal protection by Zoltán S. Zádori; Nashwan Shujaa; László Köles; Kornél P. Király; Kornélia Tekes; Klára Gyires (2257-2265).
Nociceptin (N/OFQ) and nocistatin (NST) are two endogenous neuropeptides derived from the same precursor protein, preproN/OFQ. The aim of the present work was to study the effect of NST on the ethanol-induced mucosal damage compared with that of N/OFQ following intracerebroventricular (i.c.v.) administration in the rat and to analyze the mechanism of the gastroprotective action. It was found that both NST and N/OFQ reduced the mucosal lesions in the same dose range (0.2–1 nmol i.c.v.), but in higher doses (2–5 nmol i.c.v.) the gastroprotective effect of both peptides was highly diminished. The gastroprotective effect of N/OFQ (1 nmol), but not that of NST (1 nmol), was reduced by the selective nociceptin receptor antagonist J-113397 (69 nmol i.c.v.). Similarly, decrease of the gastroprotective effect was observed after the combination of NST (1 nmol) with N/OFQ (0.6 or 1 nmol). However, addition of the gastroprotective effects was observed, when lower dose (0.2 nmol) of NST was given prior to N/OFQ (0.6 nmol). The gastroprotective effect of both N/OFQ and NST was antagonized by naloxone (27 nmol), β-funaltrexamine (20 nmol), naltrindole (5 nmol) and norbinaltorphimine (14 nmol), the μ-, δ- and κ-opioid receptor antagonists, respectively, given i.c.v. The mucosal protection was significantly decreased after bilateral cervical vagotomy. The present findings suggest that NST similar to N/OFQ, may also induce gastric mucosal protective action initiated centrally in a vagal-dependent mechanism. Opioid component is likely to be involved in the gastroprotective effect of both NST and N/OFQ.
Keywords: Gastric mucosal damage; Nociceptin; Nocistatin; Opioid antagonists; Rat;

Lack of tolerance and morphine-induced cross-tolerance to the analgesia of chimeric peptide of Met-enkephalin and FMRFa by Kshitij Gupta; Ishwar Dutt Vats; Yogendra Kumar Gupta; Kishwar Saleem; Santosh Pasha (2266-2275).
Chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFa-YFa), a κ-opioid receptor specific peptide, did not induce tolerance and cross-tolerance effects to its analgesic action on day 5 after pretreatment with either YFa or morphine for 4 days. However, pretreatment with YFa for 4 days led to the development of cross-tolerance to the analgesic effects of morphine and also 4 days of pretreatment of morphine resulted in the expression of tolerance to its own analgesic effects. Similar expression of tolerance and cross-tolerance were also observed when YFa was compared with the κ receptor agonist peptide dynorphin A(1–13) [DynA(1–13)]. Cross-tolerance effects between YFa and DynA(1–13) analgesia were also not observed on day 5. Interestingly, when YFa and DynA(1–13) were tested for their analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–13) whereas YFa maintained its analgesia for 5 days. Thus, chimeric peptide YFa may serve as a useful probe to understand pain modulation and expression of tolerance and cross-tolerance behavior with other opioids.
Keywords: Tolerance; Cross-tolerance; Chimeric peptide (YFa); Dynorphin A(1–13); Analgesia;

Two peptide transmitters co-packaged in a single neurosecretory vesicle by Elvin A. Woodruff; Kendal Broadie; Hans-Willi Honegger (2276-2280).
Numerous neurosecretory cells are known to secrete more than one peptide, in both vertebrates and invertebrates. These co-expressed neuropeptides often originate from differential cleavage of a single large precursor, and are then usually sorted in the regulated pathway into different secretory vesicle classes to allow separable release dynamics. Here, we use immuno-gold electron microscopy to show that two very different neuropeptides (the nonapeptide crustacean cardioactive peptide (CCAP) and the 30 kDa heterodimeric bursicon) are co-packaged within the same dense core vesicles in neurosecretory neurons in the abdominal ganglia of Periplaneta americana. We suggest that this co-packaging serves a physiological function in which CCAP accelerates the distribution of bursicon to the epidermis after ecdysis to regulate sclerotization of the newly formed cuticle.

Intracerebroventricular infusion of neuropeptide Y increases glucose dependent-insulinotropic peptide secretion in the fasting conscious dog by Maria P. Yavropoulou; Kalliopi Kotsa; Isaak Kesisoglou; Olympia Anastasiou; John G. Yovos (2281-2285).
The rapid increase of incretins glucose-dependent insulinotropic peptide (GIP) and glucagon like peptide-1 (GLP-1), within 5–15 min, after food ingestion, suggests that a neural mechanism might be involved in the regulation of their secretion. The aim of this study is to determine whether intracerebroventricular (i.c.v) administration of neuropeptide Y (NPY), a widely distributed neurotransmmiter, can mediate this neural regulation of GIP secretion after food consumption. Six healthy mongrel dogs were utilized for this study. A prototype epicranial apparatus was placed surgically, allowing easy and exact localization of the third ventricle for infusions or sampling. Simultaneous blood sampling was obtained from cannulation of a hind limb vein. Plasma insulin, and GIP concentrations were measured after i.c.v infusion of 5, 10 and 25 μg of NPY dissolved in 0.5 ml of artificial cerebrospinal fluid (a CSF). The secretion of GIP and insulin were increased after the injection of NPY in a different pattern. Our data indicate that NPY might be involved in a possible neural control mechanism of GIP secretion after food consumption.

Anxiolytic-like effect of neuropeptide S in the rat defensive burying by Giovanni Vitale; Monica Filaferro; Valentina Ruggieri; Sonia Pennella; Claudio Frigeri; Anna Rizzi; Remo Guerrini; Girolamo Calò (2286-2291).
Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.

Endogenous opiates and behavior: 2007 by Richard J. Bodnar (2292-2375).
This paper is the thirtieth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2007 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.
Keywords: Opioids; Enkephalins; Endorphin; Nociceptin; Endomorphins; Mu receptor; Delta receptor; Kappa receptor; ORL-1 receptor;