Peptides (v.32, #12)

Structural insight into the role of the human melanocortin 3 receptor cysteine residues on receptor function by Yingkui Yang; Min Chen; David McPherson; Vinod Mishra; Carroll M. Harmon (2377-2383).
► We examined the role of cysteine residues in melanocortin-3 receptor on receptor function. ► The mutations, C305S, C311S, and C313S significantly decrease receptor expression but C115S and C162S decrease receptor function. ► We conclude that these five cysteine residues are crucial for receptor function.Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.
Keywords: MSH; MC3R; Cysteine; MCR; GPCR;

► CRF and restraint activate CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov. ► The involvement manner of CRF receptors in the stimuli-induced activation of neurons differs with respect to the brain areas. ► The non-CRF1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp.Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF1) and CRF2 receptor (CRF2). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF1 and CRF2, are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF1- and CRF2-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60 min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF1 and/or CRF2. However, the manner of involvement for CRF1 and CRF2 in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.
Keywords: CRF; CRF receptor; Stress; PVN; Amygdala; BNST;

► Rats treated with EA showed significantly decreased food intake and reduced body weight compared with the rats in DIO and restraint group. ► EA treatment increased peptide levels of α-MSH and mRNA levels of its precursor POMC in the arcuate nuclear of hypothalamus (ARH) neurons. ► In addition, the cerebral spinal fluid (CSF) content of α-MSH was elevated by EA application. ► ARH lesions by monosodium glutamate abolished the inhibition effect of EA on food intake and body weight. ► A non-acupoint stimulation did not show the benefit effect on food intake inhibition and body weight reduction compared with restraint and ST36/SP6 EA treatment.Obesity is a major health problem in the world. Since effective remedies are rare, researchers are trying to discover new therapies for obesity, and acupuncture is among the most popular alternative approaches. This study investigated the anti-obesity mechanisms of EA, using a rat model of diet-induced obesity. After feeding with a high-fat diet for 9 weeks, a number of rats who gained weight that surpassed the maximal body weight of rats in the chow-fed group were considered obese and employed in the study. A 2 Hz EA treatment at the acupoints ST36/SP6 with the intensity increasing stepwise from 0.5–1–1.5 mA was given once a day for 30 min. Rats treated with EA showed significantly decreased food intake and reduced body weight compared with the rats in DIO and restraint group. EA treatment increased peptide levels of α-MSH and mRNA levels of its precursor POMC in the arcuate nuclear of hypothalamus (ARH) neurons. In addition, the cerebral spinal fluid (CSF) content of α-MSH was elevated by EA application. ARH lesions by monosodium glutamate abolished the inhibition effect of EA on food intake and body weight. A non-acupoint stimulation did not show the benefit effect on food intake inhibition and body weight reduction compared with restraint and ST36/SP6 EA treatment. We concluded that EA treatment at ST36/SP6 acted through ARH to significantly inhibit food intake and body weight gain when fed a high-fat diet and that the stimulation of α-MSH expression and release might be involved in the mechanism.
Keywords: Diet-induced obesity; Electroacupuncture; Arcuate nucleus of hypothalamus; POMC; α-MSH;

Display Omitted► Folic acid plus neuropeptide Y (intra-lateral septal nuclei) are antidepressant-like. ► 17β-Estradiol plus neuropeptide Y (intra-LSN) are antidepressant-like. ► Fluoxetine plus neuropeptide Y (intra-LSN) are antidepressant-like. ► These antidepressant-like effects were canceled by one NPY Y1 receptor antagonist. ► Serotonergic antidepressants interact with NPY Y1 receptors in the lateral septum.Folic acid is antidepressant, either alone or combined with several antidepressant drugs. However, the antidepressant-like actions of folic acid combined with intra-lateral septal (LSN) infusions of neuropeptide Y (NPY) in the forced swimming test (FST) have not been tested before. Thus, systemic injections of fluoxetine (20.0 mg/kg, P  < 0.05; s.c.) or 17-β estradiol (10.0 μg/rat, P  < 0.05; s.c.) or oral administrations of folic acid (50.0 mg/kg, P  < 0.05; 75.0 mg/kg, P  < 0.05) or NPY intra-LSN (3.0 μg, P  < 0.05; 3.5 μg, P  < 0.05) reduced immobility of ovariectomized Wistar rats. Subthreshold doses of: folic acid (25.0 mg/kg) or 17-β estradiol (5.0 μg/rat, P  < 0.05) or fluoxetine (15.0 mg/kg, P  < 0.05; s.c.) combined with subthreshold doses of NPY (2.5 μg/rat, P  < 0.05; intra-LSN) and these combinations produced antidepressant-like actions; which were canceled by BIBP 3226 (a NPY-Y1 receptor antagonist). It is concluded that folic acid produced antidepressant-like effects probably through the participation of the NPY Y1 receptors found in the lateral septal nuclei.
Keywords: Antidepressant; Estradiol; Folic acid; Forced swimming test; Neuropeptide Y;

Pharmacological characteristics of endokinin C/D-derived peptides in nociceptive and inflammatory processing in rats by Rumi Naono-Nakayama; Natsuki Sunakawa; Tetsuya Ikeda; Osamu Matsushima; Toshikazu Nishimori (2407-2417).
► Effect of [d-Trp8]-EKC/D or [d-Trp10]-EKC/D was longer than [d-Trp8,10]-EKC/D. ► Effect of [d-Trp10]-EKC/D depended on the number of amino acids in the N-terminal. ► [d-Trp10]-EKC/D had anti-inflammatory and anti-nociceptive effects. ► Effects of intrathecal and intradermal administration of [d-Trp10]-EKC/D were similar.Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp8]-EKC/D and [d-Trp10]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp8,10]-EKC/D and EKC/D without d-Trp disappeared after 1 h. Furthermore, the inhibitory effect of [d-Trp10]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D.
Keywords: Endokinin C/D; d-Trp; Scratching; Thermal hyperalgesia; Formalin test; Inflammation; c-Fos;

A novel immunosuppressory peptide originating from the ubiquitin sequence by Paweł Pasikowski; Tomasz Goździewicz; Piotr Stefanowicz; Jolanta Artym; Michał Zimecki; Zbigniew Szewczuk (2418-2427).
► Selected ubiquitin-derived peptides were tested for their immunomodulatory potency. ► Peptide VKTLTGKTI possessed the highest immunosuppressive potency in vitro. ► VKTLTGKTI was also active in allograft survival experiments in mice. ► Basing on ubiquitin crystal structure we designed analogs of selected peptides. ► Immunomodulatory activity of synthesized analogs (cyclic and bivalent) was tested.Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.
Keywords: Allograft skin survival; Antibody production; Cryptides; Immunosuppressory peptides; Ubiquitin fragments;

► The dose–response curve of EKA/B is “U”-shaped. ► Co-injection of EKA/B with EM-1 or EKC/D shows a depressor effect similar to that of EKA/B alone. ► Co-injection of EKC/D and EM-1 causes a dose-dependent depressor effect. ► SR140333B, SR48968C, SR142801, naloxone, and l-NAME were used for mechanism studies. ► The depressor effect of EM-1 could be fully blocked not only by naloxone but also by SR140333B, SR48968C, and SR142801.Endokinins are four novel human tachykinins, including endokinins A (EKA), B (EKB), C (EKC), and D (EKD). Endokinin A/B (EKA/B) is the common C-terminal decapeptide in EKA and EKB, while endokinin C/D (EKC/D) is the common C-terminal duodecapeptide in EKC and EKD. In this study, we attempted to investigate the interactions between EKA/B, EKC/D, and endomorphin-1 (EM-1) on the depressor effect at peripheral level. The effects of EKA/B produced a U-shaped curve. The maximal effect was caused by 10 nmol/kg. EKC/D and EM-1 showed a dose-dependent relationship. Co-administration of EKA/B (0.1, 1, 10 nmol/kg) with EM-1 produced effects similar to those of EKA/B alone but slightly lower. Co-injection of EKA/B (100 nmol/kg) with EM-1 caused an effect stronger than any separate injection. Co-administration of EKC/D (10 nmol/kg) with EM-1 (30 nmol/kg) caused a depressor effect, which was one of the tradeoffs of EM-1 and EKC/D. Mechanism studies showed that SR140333B could block the depressor effects of EKA/B, EKC/D, EM-1, EKA/B + EM-1, and EKC/D + EM-1; SR48968C could block EM-1, EKA/B, EKC/D, and EKC/D + EM-1 and partially block EKA/B + EM-1; SR142801 could block EM-1, EKC/D, and EKC/D + EM-1 and partially block EKA/B and EKA/B + EM-1; naloxone could block EM-1, EKC/D, and EKC/D + EM-1 and partially block EKA/B and EKA/B + EM-1. Pretreatment with NG-nitro-l-arginine methyl ester partially decreased depressor intensity and half-recovery time of EKA/B and EKC/D.
Keywords: Endokinin A/B; Endokinin C/D; Endomorphin-1; SR140333B; SR48968C; SR142801; NG-nitro-l-arginine methyl ester;

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells by Li-Yan Wang; Dong-Liang Zhang; Jun-Fang Zheng; Yu Zhang; Qi-Dong Zhang; Wen-Hu Liu (2436-2443).
Display Omitted► Transwell dishes were used to establish the endothelial barrier. VSMCs were under the endothelial barrier in the transwell system. ► Apelin-13 can pass through the ADMA-damaged endothelial barrier. ► Phosphorylation of MLC in VSMCs was increased by the leakage of apelin-13.Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.
Keywords: Asymmetric dimethylarginine; Apelin; Endothelial cell; Vascular smooth muscle cell; Myosin light chain; Uremia;

Blood pressure modulation following activation of mast cells by cationic cell penetrating peptides by Maamoun Basheer; Herzl Schwalb; Irit Shefler; Lilia Levdansky; Yoseph A. Mekori; Raphael Gorodetsky (2444-2451).
► Intravenous administration of cationic cell penetrating peptides (CPP) have deleterious consequences due to their induced BP drop. ► A wide range of concentrations of the cationic peptides penetratin and transportan significantly dropped blood pressure in a dose dependent manner. ► The effect was found to be mediated by mast cell activation and degranulation. ► Mast cells stabilizers inhibited this effect. ► This drawback should be considered in planning incorporation of CPP in drug delivery systems.Short cell penetrating peptides (CPP) are widely used in vitro to transduce agents into cells. But their systemic effect has not been yet studied in detail. We studied the systemic effect of the cell penetrating peptides, penetratin, transportan and pro-rich, on rat hemodynamic functions. Intra-arterial monitoring of blood pressure showed that injection of the positively charged penetratin and transportan in a wide range of concentrations (2.5–320 μg/kg) caused highly significant transient decrease in the systolic and diastolic blood pressure in a dose dependent manner (p  < 0.01). Pretreatment with histamine receptors blockers or with cromolyn, a mast cell stabilizing agent, significantly attenuated this effect. Furthermore, in vitro incubation of these both peptides with mast cells line, LAD2, caused a massive mast cell degranulation. In vitro studies showed that these CPP in a wide range of concentrations were not cytotoxic without any effect on the survival of LAD2 mast cell line. In contrast, the less positively charged and proline-rich CPP, pro-rich, had no systemic effects with no effect on mast cell degranulation. Our results indicate that intravenously administrated positively charged CPP may have deleterious consequences due to their induced BP drop, mediated by mast cell activation. Therefore, the major effect of mast cell activation on BP should be considered in developing possible future drug therapies based on the injection of membrane-permeable and positively charged CPP. Nevertheless, lower levels of such CPP may be considered as a treatment of systemic high BP through moderate systemic mast cell activation.
Keywords: Cell penetrating peptide; Penetratin; Transportan; Pro-rich; Hypotension; Histamine; Serotonin; Mast cell;

Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts by Yong-Gang Zhang; Ze-Jian Kuang; Yan-Yan Mao; Rui-Hong Wei; Shi-Lin Bao; Li-Biao Wu; Yu-Guang Li; Chao-Shu Tang (2452-2458).
► Antisense oligonucleotides of osteopontin inhibited urotensin II (UII)-induced migration of adventitial fibroblasts. ► UII promoted osteopontin mRNA expression and secretion from adventitial fibroblasts. ► Inhibition of UT, Ca2+ channel, protein kinase C, calcineurin, mitogen-activated protein kinase and Rho kinase signal transduction pathways attenuated osteopontin expression stimulated by UII.Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P  < 0.01) but not sense or mismatch oligonucleotides (P  > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10−8  mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P  < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10−6  mol/l), and Ca2+ channel blocker nicardipine (10−5  mol/l), protein kinase C (PKC) inhibitor H7 (10−5  mol/l), calcineurin inhibitor cyclosporine A (10−5  mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10−5  mol/l) and Rho kinase inhibitor Y-27632 (10−5  mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca2+ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways.
Keywords: Osteopontin; Urotensin II; Migration; Adventitial fibroblasts; Signal transduction; Peptide;

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart by Afshin Nazari; Seyed Shahabeddin Sadr; Mahdieh Faghihi; AliReza Imani; Maryam Moghimian (2459-2466).
► The dose–response curve of AVP is U-shaped. ► Administration of exogenous AVP shows a cardioprotective effect against ischemia/reperfusion injury. ► Cardioprotective effect of AVP was partially blocked by SR49059 as AVP-selective V1 receptor antagonist.The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups (n  = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.
Keywords: Ischemia; Reperfusion; Vasopressin; Preconditioning; Heart;

Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria by Shan Gao; Kuichang Yuan; Amin Shah; Jong Suk Kim; Woo Hyun Park; Suhn Hee Kim (2467-2473).
► N-acetyl cystein, α-lipoic acid and tempol inhibited high pacing frequency-induced ANP secretion. ► NADPH oxidase activity was increased by high pacing stimulation and in diabetic rat atria. ► In diabetic atria, the response of ANP secretion to high stimulation frequency attenuated but the response to NAC and apocynin augmented.Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H2O2 level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.
Keywords: ROS; NADPH oxidase; Antioxidant; Atrial natriuretic peptide; Frequency; Diabetes;

The apelinergic system in the developing lung: Expression and signaling by Paulina Piairo; Rute S. Moura; Cristina Nogueira-Silva; Jorge Correia-Pinto (2474-2483).
► Apelin and APJ are constitutively expressed in the developing lung. ► Fetal apelin expression is higher than adult expression. ► APJ exhibits nuclear localization in pulmonary epithelium. ► Apelin's inhibitory effect on fetal lung growth is associated with inhibition of p38 and JNK signaling pathways.Apelin and its receptor APJ constitute a signaling pathway best recognized as an important regulator of cardiovascular homeostasis. This multifunctional peptidergic system is currently being described to be involved in embryonic events which extend into vascular, ocular and heart development. Additionally, it is highly expressed in pulmonary tissue. Therefore, the aim of this study was to investigate the role of apelinergic system during fetal lung development. Immunohistochemistry and Western blot analysis were used to characterize apelin and APJ expression levels and cellular localization in normal fetal rat lungs, at five different gestational ages as well as in the adult. Fetal rat lung explants were cultured in vitro with increasing doses of apelin. Treated lung explants were morphometrically analyzed and assessed for MAPK signaling modifications. Both components of the apelinergic system are constitutively expressed in the developing lung, with APJ exhibiting monomeric, dimeric and oligomeric forms in the pulmonary tissue. Pulmonary epithelium also displayed constitutive nuclear localization of the receptor. Fetal apelin expression is higher than adult expression. Apelin supplementation inhibitory effect on branching morphogenesis was associated with a dose dependent decrease in p38 and JNK phosphorylation. The results presented provide the first evidence of the presence of an apelinergic system operating in the developing lung. Our findings also suggest that apelin inhibits fetal lung growth by suppressing p38 and JNK signaling pathways.
Keywords: Apelin; APJ; Lung development; MAP kinases;

Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates by Arianna Tavanti; Giuseppantonio Maisetta; Gaetano Del Gaudio; Raffaele Petruzzelli; Maurizio Sanguinetti; Giovanna Batoni; Sonia Senesi (2484-2487).
Candida glabrata infections are difficult to eradicate due to the low susceptibility to azoles. ► C. glabrata exhibits scarce susceptibility to cationic antimicrobial peptides. ► The human cationic peptide hepcidin 20 shows fungicidal activity toward C. glabrata isolates. ► Fungicidal effect of the Hep-20 is potentiated at acidic pH. ► A synergistic effect was observed for Hep-20 in combination with common antifungals. Candida glabrata infections are often difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been shown to be promising novel therapeutic candidates for the treatment of fungal infection. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. Interestingly, all isolates were susceptible to Hep-20 (100–200 μg/ml) at pH 7.4, whereas the fungicidal effect of the peptide was higher (50 μg/ml) at acidic pH values. In addition, an increased antifungal activity was observed for Hep-20 with amphotericin B and a synergistic effect was demonstrated for the Hep-20/fluconazole and Hep-20/caspofungin combinations.
Keywords: Candida glabrata; Human hepcidin 20; Antimicrobial peptides; Antifungal activity; Synergistic effect;

► Human high molecular weight kininogen binds to Candida albicans cell wall proteins. ► Als3 adhesin belongs to major high molecular weight fungal binders of kininogen. ► Enolase, phosphoglycerate mutase and triosephosphate isomerase also bind kininogen. ► Cell-wall binding region on kininogen comprises residues 479–564 of domains 5 and 6.An excessive production of vasoactive and proinflammatory bradykinin-related peptides, the kinins, is often involved in the human host defense against microbial infections. Recent studies have shown that a major fungal pathogen to humans, Candida albicans, can bind the proteinaceous kinin precursor, the high molecular weight kininogen (HK) and trigger the kinin-forming cascade on the cell surface. In this work, we preliminarily characterized a molecular mechanism underlying the HK adhesion to the fungal surface by (i) identification of major kininogen-binding constituents on the candidial cell wall and (ii) mapping the cell wall-binding regions on HK molecule. A major fraction of total fungal kininogen-binding capacity was assigned to β-1,3-glucanase-extractable cell wall proteins (CWP). By adsorption of CWP on HK-coupled agarose gel and mass spectrometric analysis of the eluted material, major putative HK receptors were identified, including Als3 adhesin and three glycolytic enzymes, i.e., enolase 1, phosphoglycerate mutase 1 and triosephosphate isomerase 1. Using monoclonal antibodies directed against selected parts of HK molecule and synthetic peptides with sequences matching selected HK fragments, we assigned the major fungal cell wall-binding ability to a short stretch of amino acids in the C-terminal part of domain 3 and a large continuous region involving the C-terminal part of domain 5 and N-terminal part of domain 6 (residues 479–564). The latter characteristics of HK binding to C. albicans surface differ from those reported for bacteria and host cells.
Keywords: Candida albicans cell wall; Contact system; Kinins; Agglutinin-like sequence; Enolase; Phosphoglycerate mutase;

Structure–activity relationships of a snake cathelicidin-related peptide, BF-15 by Wei Chen; Baowei Yang; Huimin Zhou; Lidan Sun; Jie Dou; Hai Qian; Wenlong Huang; Yicheng Mei; Jing Han (2497-2503).
► C-terminal amidated analogs of BF-15 were designed and synthesized. ► Antibacterial activity and haemolytic activity of the analogs were identified. ► Analog B1 showed high antibacterial activity and low haemolytic activity. ► The antibacterial mechanism of the analog B1 was delineated.Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.
Keywords: Snake cathelicidin; Antimicrobial peptides; Cationic; Amphipathic; Helical;

Design and synthesis of biologically active peptides: A ‘tail’ of amino acids can modulate activity of synthetic cyclic peptides by Alberto Bryan; Leroy Joseph; James A. Bennett; Herbert I. Jacobson; Thomas T. Andersen (2504-2510).
Display Omitted► Activity of cyclic peptides can be modified by addition of amino acids outside of the ring, i.e., by adding a tail. ► Homobiotic peptides are biosuperior to toxic xenobiotic molecules. ► AFPep analogs with a hydrophobic tail yield enhanced activity relative to AFPep. ► AFPep analogs with a hydrophilic tail yield decreased activity relative to AFPep. ► An analog with a disrupted pharmacophore and with a hydrophilic tail is an antagonist.In earlier work, we synthesized a cyclic 9-amino acid peptide (AFPep, cyclo[EKTOVNOGN]) and showed it to be useful for prevention and therapy of breast cancer. In an effort to explore the structure–function relationships of AFPep, we have designed analogs that bear a short ‘tail’ (one or two amino acids) attached to the cyclic peptide distal to its pharmacophore. Analogs that bore a tail of either one or two amino acids, either of which had a hydrophilic moiety in the side chain (e.g., cyclo[EKTOVNOGN]FS) exhibited greatly diminished biological activity (inhibition of estrogen-stimulated uterine growth) relative to AFPep. Analogs that bore a tail of either one or two amino acids which had hydrophobic (aliphatic or aromatic) side chains (e.g., cyclo[EKTOVNOGN]FI) retained (or had enhanced) growth inhibition activity. Combining in the same biological assay a hydrophilic-tailed analog with either AFPep or a hydrophobic-tailed analog resulted in decreased activity relative to that for AFPep or for the hydrophobic-tailed analog alone, suggesting that hydrophilic-tailed analogs are binding to a biologically active receptor. An analog with a disrupted pharmacophore (cyclo[EKTOVGOGN]) exhibited little or no growth inhibition activity. An analog with a hydrophilic tail and a disrupted pharmacophore (cyclo[EKTOVGOGN]FS) exhibited no growth inhibition activity of its own and did not affect the activity of a hydrophobic-tailed analog, but enhanced the growth inhibition activity of AFPep. These results are discussed in the context of a two-receptor model for binding of AFPep and ring-and-tail analogs. We suggest that tails on cyclic peptides may comprise a useful method to enhance diversity of peptide design and specificity of ligand–receptor interactions.
Keywords: Cyclic peptide; Ring-and-tail synthetic peptide; Rational design of synthetic peptide; Two-receptor model; Antagonist; Homobiotic peptide;

► We determined if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal phase and the oestrous and during the follicular phase induced with the ram effect. ► CSF was collected from ewes during spontaneous oestrous and mid-luteal phase (Experiment 1) and from ewes in non-breeding season before and after follicular phase was induced with rams (Experiment 2). ► There were no differences in MCH concentrations in CSF during the mid-luteal phase and spontaneous oestrous. ► MCH concentrations in CSF were higher 24 h after rams introduction. ► We concluded that the MCHergic system participates in the ram effect response but not in spontaneous oestrous cycles in sheep.The melanin-concentrating hormone (MCH) is a neuropeptide synthesized by neurons of the lateral hypothalamus and incerto-hypothalamic area that project throughout the central nervous system. The aims of the present report were: (1) to determine if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal and the oestrous phase of spontaneous oestrous cycles; and (2) to study if MCH levels in CSF of ewes vary acutely during the follicular phase induced with the ram effect in anoestrous ewes. In the first experiment, CSF was collected from 8 adult ewes during spontaneous oestrous and during the mid-luteal phase (8–10 days after natural oestrus). In the second experiment, performed during the mid non-breeding season, a follicular phase was induced with the ram effect. After isolating a group of 16 ewes from rams, CSF was obtained from 5 of such ewes (control group). Three rams were joined with the ewes, and samples were obtained 12 h (n  = 6) and 24 h (n  = 5) later. In Experiment 1, there were no differences in MCH concentrations in CSF measured during the mid-luteal phase and spontaneous oestrus (0.14 ± 0.04 vs. 0.16 ± 0.05 ng/mL respectively). In Experiment 2, MCH concentrations tended to increase 12 h after rams introduction (0.15 ± 0.08 vs. 0.35 ± 0.21 ng/mL, P  = 0.08), and increased significantly 24 h after rams introduction (0.37 ± 0.15 ng/mL, P  = 0.02). We concluded that MCH concentration measured in the CSF from ewes increased markedly during the response to the ram effect but not during the natural oestrous cycle of ewes.

Beyond the metabolic role of ghrelin: A new player in the regulation of reproductive function by Giampiero Muccioli; Teresa Lorenzi; Maria Lorenzi; Corrado Ghè; Elisa Arnoletti; Giuseppina Mattace Raso; Mario Castellucci; Oreste Gualillo; Rosaria Meli (2514-2521).
Ghrelin is a gastric peptide, discovered by Kojima et al. (1999) as a result of the search for an endogenous ligand interacting with the “orphan receptor” GHS-R1a (growth hormone secretagogue receptor type 1a). Ghrelin is composed of 28 aminoacids and is produced mostly by specific cells of the stomach, by the hypothalamus and hypophysis, even if its presence, as well as that of its receptors, has been demonstrated in many other tissues, not least in gonads. Ghrelin potently stimulates GH release and participates in the regulation of energy homeostasis, increasing food intake, decreasing energy output and exerting a lipogenetic effect. Furthermore, ghrelin influences the secretion and motility of the gastrointestinal tract, especially of the stomach, and, above all, profoundly affects pancreatic functions. Despite of these previously envisaged activities, it has recently been hypothesized that ghrelin regulates several aspects of reproductive physiology and pathology. In conclusion, ghrelin not only cooperates with other neuroendocrine factors, such as leptin, in the modulation of energy homeostasis, but also has a crucial role in the regulation of the hypothalamic–pituitary gonadal axis. In the current review we summarize the main targets of this gastric peptide, especially focusing on the reproductive system.
Keywords: Ghrelin; Energy homeostasis; Reproduction;

Endogenous opiates and behavior: 2010 by Richard J. Bodnar (2522-2552).
► This paper is the 33rd annual review of research on the endogenous opioid system. ► It summarizes 2010 behavioral, molecular and pharmacological opioid papers. ► Topics include opioid analgesic, stress, tolerance and dependence effects. ► Topics also include learning, ingestion, alcohol and drugs of abuse. ► Topics also examine sex, mental illness, and neurologic disorders.This paper is the thirty-third consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2010 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 (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17).
Keywords: Morphine; Naloxone; Opioid receptors; Opioid peptides;