Peptides (v.47, #C)

Intermedin enhances sympathetic outflow via receptor-mediated cAMP/PKA signaling pathway in nucleus tractus solitarii of rats by Peng Li; Hai-Jian Sun; Ying Han; Jue-Jin Wang; Feng Zhang; Chao-Shu Tang; Ye-Bo Zhou (1-6).
Direct administration of intermedin (IMD) into the brain elicits cardiovascular effects different from the systemic administration. Nucleus tractus solitarii (NTS) is an important region for the cardiovascular regulation. The present study was designed to determine the effect of IMD on modulating the sympathetic outflow and its related molecular mechanism in the NTS. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in anesthetized rats. Site-specific microinjection of IMD (20 pmol) bilaterally into the NTS significantly increased RSNA and MAP. IMD-evoked increases of RSNA and MAP were almost abolished by pretreatment with receptor antagonist ADM22-52, an adenylyl cyclase (AC) inhibitor SQ22536, or a protein kinase A (PKA) inhibitor Rp-cAMP. However, pretreatment with another receptor antagonist calcitonin gene-related peptide (CGRP)8-37 did not suppress the increases of RSNA and MAP induced by IMD. Furthermore, IMD increased the cyclic adenosine monophosphate (cAMP) level, which was inhibited by ADM22-52 pretreatment in the NTS. These results suggest that IMD participates in the sympathetic nerve activity and central regulation of the cardiovascular system and a receptor-mediated cAMP/PKA signaling pathway is involved in IMD-induced effects in the NTS.
Keywords: Intermedin; Sympathetic outflow; Protein kinase A; Nucleus tractus solitarii;

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats by Nandkishor R. Kotagale; Nikhilesh P. Paliwal; Manish M. Aglawe; Milind J. Umekar; Brijesh G. Taksande (7-11).
Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu31, Pro34]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu31, Pro34]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu31, Pro34]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.
Keywords: Agmatine; Neuropeptide Y; NPY Y1 receptors; Depression; Immobility time; FST;

Ghrelin in ocular pathophysiology: From the anterior to the posterior segment by Sara Azevedo-Pinto; Paulo Pereira-Silva; Amândio Rocha-Sousa (12-19).
Ghrelin is a 28 amino acid acylated peptide produced in several organs that binds the growth hormone secretagogues receptor type 1a (GHSR-1a). It acts over a wide range of systems, e.g. the endocrine, cardiovascular, musculoskeletal and immune systems and the eye. The aim of this work is to review the physiologic and pathologic implications of the ghrelin-GHSR-1a in the eye. A systematic revision of studies published between 2000 and 2013 in English, Spanish or Portuguese in MEDLINE, EMBASE and Scopus was performed. Search words used included: ghrelin, GHSR-1a, ocular production, iris muscular kinetics, ciliary body, glaucoma, retinopathy and uvea. The production of ghrelin by the ocular tissue has been detected both in the anterior and posterior segments, as well as the presence of GHSR-1a. This peptide promotes the relaxation of the iris sphincter and dilator muscles, being this effect independent from GHSR-1a and dependent on prostaglandins release in the first case and dependent on GHSR-1a in the second. Regarding ocular pathology, ghrelin levels in the aqueous humor appear to be decreased in individuals with glaucoma. Moreover, ghrelin has been shown to decrease the intraocular pressure in animal models of ocular hypertension through GHSR-1a. In the posterior segment, the ghrelin-GHSR-1a system interferes with the development of oxygen-induced retinopathy, being protective in the vaso-obliterative phase and deleterious in the vaso-proliferative stage of the disease. Thus, the ghrelin-GHSR-1a system presents as a possible local regulatory mechanism in the eye, with pathophysiological implications, constituting a target for future clinical and therapeutic research and interventions.
Keywords: Ghrelin; GHSR-1a; Iris muscles; Glaucoma; Retinopathy;

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as well as food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypothalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rs in the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administration of its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increases food intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-lived lasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested, however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural burrow/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraging group), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free food (exercise control). We microinjected BIIE0246 (antagonist) and PYY(3-36) (agonist) into the Arc to test the role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affected by Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rs by PYY(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing general malaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of the Arc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.
Keywords: Appetitive behavior; Siberian hamster; Peptide YY; BIIE0246; Food foraging; Food deprivation;

Swimming training prevents fat deposition and decreases angiotensin II-induced coronary vasoconstriction in ovariectomized rats by Patrick Wander Endlich; Erick Roberto Gonçalves Claudio; Washington Luiz da Silva Gonçalves; Sonia Alves Gouvêa; Margareth Ribeiro Moysés; Glaucia Rodrigues de Abreu (29-35).
We investigated the effects of chronic swimming training (ST) on the deposition of abdominal fat and vasoconstriction in response to angiotensin II (ANG II) in the coronary arterial bed of estrogen deficient rats. Twenty-eight 3-month old Wistar female rats were divided into 4 groups: sedentary sham (SS), sedentary-ovariectomized (SO), swimming-trained sham (STS) and swimming-trained ovariectomized (STO). ST protocol consisted of a continuous 60-min session, with a 5% BW load attached to the tail, completed 5 days/week for 8-weeks. The retroperitoneal, parametrial, perirenal and inguinal fat pads were measured. The intrinsic heart rate (IHR), coronary perfusion pressure (CPP) and a concentration–response curve to ANG II in the coronary bed was constructed using the Langendorff preparation. Ovariectomy (OVX) significantly reduced 17-β-estradiol plasma levels in SO and STO groups (p  < 0.05). The STO group had a significantly reduced retroperitoneal and parametrial fat pad compared with the SO group (p  < 0.05). IHR values were similar in all groups; however, baseline CPP was significantly reduced in the SO, STS and STO groups compared with the SS group (p  < 0.05). ANG II caused vasoconstriction in the coronary bed in a concentration-dependent manner. The SO group had an increased response to ANG II when compared with all other experimental groups (p  < 0.05), which was prevented by 8-weeks of ST in the STO group (p  < 0.05). OVX increased ANG II-induced vasoconstriction in the coronary vascular bed and abdominal fat pad deposition. Eight weeks of swimming training improved these vasoconstrictor effects and decreased abdominal fat deposition in ovariectomized rats.
Keywords: Estrogen deficiency; Coronary dysfunction; RAS; Physical exercise;

Angiotensin AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway by Shan Gao; Byung Mun Park; Seung Ah Cha; Woo Hyun Park; Byung Hyun Park; Suhn Hee Kim (36-44).
Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT2R are still controversial. The aim of the present study is to define the effect of AT2R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1 μM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1 μM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT2R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT1R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10 min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT2R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT2R antagonist for 10 min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT1R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT2R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.
Keywords: Angiotensin; Receptor; Atrial natriuretic peptide; Pressure; Hypertension;

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.
Keywords: Ghrelin; GHSR; GFP; Kisspeptin; AgRP; Reproduction;

Gamma2-melanocyte-stimulating hormone (γ2MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle3, D-Phe6]-γ2MSH (NDP-γ2MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ2MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ2MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ2MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ2MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.
Keywords: Kidney; Sodium chloride; Gamma-MSH; Natriuresis; Blood pressure; Cell culture;

An effective technique for the processing of saliva for the analysis of leptin and adiponectin by S. Thanakun; H. Watanabe; S. Thaweboon; Y. Izumi (60-65).
The recovery of protein from saliva has been extensively investigated as a method to monitor health. The aim of this study was to compare filtration and centrifugation as two methods of saliva processing necessary for determining the levels of salivary leptin and adiponectin. Thirty-seven healthy patients (median age of 45 years; range 35–73) participated in the study. Unstimulated whole saliva was collected by a drooling technique. An aliquot was filtered using a Millex-Millipore® (0.45 μm PVDF Dura Pore membrane) syringe and a second aliquot was centrifuged at 15 000 ×  g for 15 min at 4 °C. Leptin and adiponectin levels were analyzed using an ELISA kit for serum (RayBio®, GA, USA) with minor modifications. Leptin and adiponectin levels following the filtration technique yielded comparable results with those after centrifugation. Correlation was observed between filtered and centrifuged salivary leptin levels ((r  = 0.9155; 95% CI 0.8362–0.9573; p  < 0.0001) with concordance correlation coefficient k 0.9114 (95% CI 0.8332–0.9539)). Less correlation was observed for adiponectin ((r  = 0.5718; 95% CI 0.3041–0.7558; p  = 0.0002) with concordance correlation coefficient k 0.5586 (95% CI 0.2977–0.7419)). Using a Bland–Altman plot, similar measurements for both adipocytokines were observed with mean difference within a 95% CI, and interpreted as no systematic differences between the two processing techniques. This study showed that filtration is an alternative saliva processing technique to retrieve supernatant for protein analysis. Filtered saliva yielded leptin and adiponectin concentrations comparable with those obtained from centrifuged saliva.
Keywords: Saliva; Processing; Centrifugation; Filtration; Leptin; Adiponectin;

Copeptin, adropin and irisin are polypeptide hormones implicated in energy homostasis and diabetes. The purposes of this study were (1) to compare the copeptin, adropin and irisin concentrations between colostrum, transitional and mature milk and plasma in lactating women with and without GDM and (2) to compare these values with those from non-lactating women. Venous blood samples were obtained before suckling from 15 healthy lactating women aged 26–30 years, 15 lactating women with GDM aged 26–32 years, and 14 age-matched controls aged 25–31 years. Colostrum, transitional milk and mature milk samples were collected just before suckling. The concentration of copeptin was determined by EIA while the concentrations of adropin and irisin were determined by ELISA. The levels of copeptin, adropin and irisin in the colostrum were significantly higher than those in transitional and mature milk samples from healthy women; also, transitional milk had higher copeptin, adropin and irisin concentrations than mature milk. The amounts of copeptin in the colostrum and transitional milk were significantly higher than in mature milk samples from women with GDM, while the amounts of adropin and irisin were significantly lower. The relative concentrations of copeptin, adropin and irisin in the plasma samples from these groups of women were similar to those in the colostrum, transitional and mature milk samples, but the latter concentrations were higher than those in the plasma. These peptides could influence the regulation of metabolic pathways and the postnatal growth and development of different organs in the newborn.
Keywords: Copeptin; Adropin; Irisin; Breast milk; Gestational diabetes mellitus;

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms by Tim Ruder; Syed Abid Ali; Kiel Ormerod; Andreas Brust; Mary-Louise Roymanchadi; Sabatino Ventura; Eivind A.B. Undheim; Timothy N.W. Jackson; A. Joffre Mercier; Glenn F. King; Paul F. Alewood; Bryan G. Fry (71-76).
It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.
Keywords: Octopus; Tachykinin; Venom; Peptide;

The effects of angiotensin II signaling pathway in the systolic response to acute stretch in the normal and ischemic myocardium by João Sérgio Neves; Ricardo Castro-Ferreira; Ricardo Ladeiras-Lopes; Manuel Neiva-Sousa; André M. Leite-Moreira; João Almeida-Coelho; Ricardo Fontes-Carvalho; João Ferreira-Martins; Adelino F. Leite-Moreira (77-84).
Acute myocardial stretch elicits a biphasic increase in contractility: an immediate increase, known as Frank–Starling mechanism (FSM), followed by a progressive increase, regarded as slow force response (SFR). In this study, we characterized the contractile response to acute stretch from 92 to 100% L max in rabbit papillary muscles (n  = 86) under normoxic and ischemic conditions, and its modulation by angiotensin II signaling pathway. Under normoxia, the FSM was independent of Na+/H+-exchanger, reverse mode of Na+/Ca2+-exchanger (r-NCX), AT1 receptor, AT2 receptor and PKC. Regarding the SFR, it was mediated by AT1 receptor activation and its downstream effectors PKC, Na+/H+-exchanger and r-NCX. Ischemia negatively impacted on the FSM and abolished the SFR, with the muscles exhibiting a time-dependent decline in contractility. Under ischemic conditions, FSM was not influenced by AT1 and AT2 receptors or PKC activation. AT1 receptor antagonism rescued the progressive deterioration in contractility, an effect partially dependent on AT2 receptor activation.
Keywords: Angiotensin II; Myocardial stretch; Frank–Starling mechanism; Slow force response; Ischemia;

The interface of hypothalamic–pituitary–adrenocortical axis and circulating brain natriuretic peptide in prediction of cardiopulmonary performance during physical stress by Dejana Popovic; Bojana Popovic; Bosiljka Plecas-Solarovic; Vesna Pešić; Vidan Markovic; Stanimir Stojiljkovic; Vladan Vukcevic; Ivana Petrovic; Marko Banovic; Milan Petrovic; Bosiljka Vujisic-Tesic; Miodrag C. Ostojic; Arsen Ristic; Svetozar S. Damjanovic (85-93).
Brain natriuretic peptide (NT-pro-BNP) was implicated in the regulation of hypothalamic–pituitary–adrenocortical (HPA) responses to psychological stressors. However, HPA axis activation in different physical stress models and its interface with NT-pro-BNP in the prediction of cardiopulmonary performance is unclear. Cardiopulmonary test on a treadmill was used to assess cardiopulmonary parameters in 16 elite male wrestlers (W), 21 water polo player (WP) and 20 sedentary age-matched subjects (C). Plasma levels of NT-pro-BNP, cortisol and adrenocorticotropic hormone (ACTH) were measured using immunoassay sandwich technique, radioimmunoassay and radioimmunometric techniques, respectively, 10 min before test (1), at beginning (2), at maximal effort (3), at 3rd min of recovery (4). In all groups, NT-pro-BNP decreased between 1 and 2; increased from 2 to 3; and remained unchanged until 4. ACTH increased from 1 to 4, whereas cortisol increased from 1 to 3 and stayed elevated at 4. In all groups together, ΔNT-pro-BNP2/1 predicted peak oxygen consumption (B  = 37.40, r  = 0.38, p  = 0.007); cortisol at 3 predicted heart rate increase between 2 and 3 (r  = −0.38,B  = −0.06, p  = 0.005); cortisol at 2 predicted peak carbon-dioxide output (B  = 2.27, r  = 0.35, p  < 0.001); ΔACTH3/2 predicted peak ventilatory equivalent for carbon-dioxide (B  = 0.03, r  = 0.33, p  = 0.003). The relation of cortisol at 1 with NT-pro-BNP at 1 and 3 was demonstrated using logistic function in all the participants together (for 1/cortisol at 1 B  = 63.40, 58.52; r  = 0.41, 0.34; p  = 0.003, 0.013, respectively). ΔNT-pro-BNP2/1 linearly correlated with ΔACTH4/3 in WP and W (r  = −0.45, −0.48; p  = 0.04, 0.04, respectively). These results demonstrate for the first time that HPA axis and NT-pro-BNP interface in physical stress probably contribute to integrative regulation of cardiopulmonary performance.
Keywords: Brain natriuretic peptide; Cortisol; Adrenocorticotropic hormone; Hypothalamic–pituitary–adrenocortical axis; Cardiopulmonary test; Stress axis;

Involvement of orexin-2 receptors in the ventral tegmental area and nucleus accumbens in the antinociception induced by the lateral hypothalamus stimulation in rats by Hassan Azhdari-Zarmehri; Zahra Reisi; Anoumid Vaziri; Amir Haghparast; Pariya Shaigani; Abbas Haghparast (94-98).
Orexin, which is mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), plays an important role in pain modulation. Both kinds of orexin-1 (Ox1) and orexin-2 (Ox2) receptors have been found at high density in the ventral tegmental area (VTA) and nucleus accumbens (NAc). However, the quantity of Ox1 receptors in the VTA is more than that in the NAc. Additionally, it seems that the functional interaction between the LH, VTA and NAc implicates pain processing and modulation. In this study, we tried to examine the involvement of Ox2 receptors in the NAc and VTA using tail-flick test as an animal model of acute pain following microinjection of effective dose of carbachol (125 nmol/0.5 μl saline) into the LH. In this set of experiments, different doses of TCS OX2 29 as an Ox2 receptor antagonist were microinjected into the VTA (1, 7 and 20 nmol/0.3 μl DMSO) and the NAc (2, 10, 20 and 40 nmol/0.5 μl DMSO) 5 min prior to carbachol administration. Administration of TCS OX2 29 into the VTA and NAc dose-dependently blocked intra-LH carbachol-induced antinociception. However, the inhibitory effect of TCS OX2 29 as an Ox2 receptor antagonist was more potent in the VTA than that in the NAc. It seems that VTA orexinergic receptors are more effective on LH stimulation-induced antinociception and the modulation of pain descending inhibitory system originated from the LH than those of the same receptors in the nucleus accumbens in rats.
Keywords: Orexin-2 receptor; Lateral hypothalamus; Ventral tegmental area; Nucleus accumbens; Antinociception; Rat;

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2 by Ying Nie; Monica G. Ferrini; Yanjun Liu; Adrian Anghel; Enma V. Paez Espinosa; Ronald C. Stuart; Kabirullah Lutfy; Eduardo A. Nillni; Theodore C. Friedman (99-109).
The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.
Keywords: Post-translational processing; Opioids; Endorphin; Prohormone convertase; Pituitary; Drug addiction;

C-type natriuretic peptide transcriptomic profiling increases in human leukocytes of patients with chronic heart failure as a function of clinical severity by M. Cabiati; L. Sabatino; R. Caruso; A. Verde; C. Caselli; T. Prescimone; D. Giannessi; S. Del Ry (110-114).
The aim of this study was to evaluate the transcriptomic profiling of C-type natriuretic peptide (CNP) and of its specific receptor, NPR-B in human leukocytes of heart failure (HF) patients as a function of clinical severity, assessing the possible changes with respect to healthy subjects (C). mRNA expression was evaluated by Real-Time PCR and total RNA was extracted from leukocytes of C (n  = 8) and of HF patients (NYHA I–II, n  = 7; NYHA III–IV, n  = 13) with PAXgene Blood RNA Kit. Significantly higher levels of CNP mRNA expression were found in HF patients as a function of clinical severity (C = 0.23 ± 0.058, NYHA I–II = 0.47 ± 0.18, NYHA III–IV = 2.58 ± 0.71, p  = 0.005 C vs NYHA III–IV, p  = 0.017 NYHA I–II vs NYHA III–IV) and NPR-B transcript levels resulted down-regulated in HF patients with higher NYHA class (C = 2.2 ± 0.61, NYHA I–II = 2.76 ± 0.46, NYHA III–IV = 0.29 ± 0.13, p  = 0.001 C vs NYHA III–IV, p  < 0.0001 NYHA I–II vs NYHA III–IV). A significant negative correlation between CNP and NPR-B mRNA expression (r  = 0.5, p  = 0.03) was also observed. These results suggest a co-regulation of NPR-B and CNP expression supporting the relevance of this receptor in human disease characterized by a marked inflammatory/immune component and suggesting the possibility of manipulating inflammation via pharmacological agents selective for this receptor.
Keywords: C-type natriuretic peptide; Natriuretic peptide receptor; Real-Time PCR; mRNA expression; Blood; Human leukocytes; Heart failure;

Alteration of sweet taste in high-fat diet induced obese rats after 4 weeks treatment with exenatide by Xiao-juan Zhang; Yu-qing Wang; Yang Long; Lei Wang; Yun Li; Fa-bao Gao; Hao-ming Tian (115-123).
Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is effective in inducing weight loss. The exact mechanisms are not fully understood. Reduced appetite and food intake may play important roles. Sweet taste contributes to food palatability, which promotes appetite. Interestingly, GLP-1 and its receptor are expressed in the taste buds of rodents and their interaction has an effect on mediating sweet taste sensitivity. Our aim was to investigate whether sweet taste will be changed after long term treatment with exenatide. The results showed that high-fat diet induced obese rats (HF-C) presented metabolic disorders in food intake, body weight, blood glucose and lipid metabolism compared with long term exenatide treated obese rats (EX) and normal chow fed control rats (NC). Meanwhile, greater preference for sweet taste was observed in HF-C rats but not in EX rats. Compared with NC rats, brain activities induced by sweet taste stimulation were stronger in HF-C rats, however these stronger activities were not found in EX rats. We further found reduced sweet taste receptor T1R3 in circumvallte taste buds of HF-C rats, while GLP-1 was increased. Besides, serum leptin was evaluated in HF-C rats with decreased leptin receptor expressed in taste buds. These changes were not observed in EX rats, which suggest them to be the underlying hormone and molecular mechanisms responsible for alterations in sweet taste of HF-C rats and EX rats. In summary, our results suggest that long term treatment with exenatide could benefit dietary obese rats partially by reversing sweet taste changes.
Keywords: Exenatide; Obesity; Taste;

The anti-inflammatory potential of neuropeptide FF in vitro and in vivo by Yu-Long Sun; Xiao-Yuan Zhang; Tao Sun; Ning He; Jing-Yi Li; Yan Zhuang; Qian Zeng; Jing Yu; Quan Fang; Rui Wang (124-132).
Neuropeptide FF (NPFF) has many functions in regulating various biological processes. However, little attention has been focused on the anti-inflammatory effect of this peptide. In the present study, the in vitro anti-inflammatory activity of NPFF in both primary peritoneal macrophages and RAW 264.7 macrophages was investigated. Our data showed that NPFF suppressed the nitric oxide (NO) production of macrophages in the inflammation process. RF9, a reported antagonist of NPFF receptors, completely blocked the NPFF-induced NO suppression, suggesting a NPFF receptors-mediated pathway is mainly involved. Down-regulation of the nitric oxide synthases significantly inhibited the NPFF-induced NO reduction, indicating the involvement of nitric oxide synthases. However, the nitric oxide synthases were not the only route by which NPFF modulated the NO levels of macrophages. Pharmacological antagonists of the NF-κB signal pathway also completely suppressed the NPFF-induced NO decline. Moreover, we also observed that NPFF is capable of blocking the LPS-induced nuclear translocation of p65 in macrophages, implying the involvement of the NF-κB signal pathway. Finally, we observed that NPFF markedly attenuated the carrageenan-induced mouse paw edema, indicating that NPFF is capable of exerting anti-inflammatory potency in vivo. Collectively, our findings reveal the potential role of NPFF in the anti-inflammatory field both in vitro and in vivo, which will be helpful for the further exploitation of NPFF utility therapeutically.
Keywords: Neuropeptide FF; Neuropeptide FF 2 receptor; Nitric oxide; Macrophage; NF-κB; Inflammation;

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock by Reginald Matejec; Friederike Kayser; Frauke Schmal; Florian Uhle; Rolf-Hasso Bödeker; Hagen Maxeiner; Julia Anna Kolbe (133-141).
Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kgbodyweight  h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.
Keywords: Proopiomelanocortin (POMC); Septic shock; Corticotropin releasing hormone (CRH); Alpha melanocyte stimulating hormone (α-MSH); Human leukocyte antigen-DR (HLA-DR); Tumor necrosis factor-α (TNF-α);

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9 ± 1.2 years, BMI: 34.2 ± 1.1 kg/m2) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60 min/day for 5 days/week at ∼85% HRmax). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40 mU/m2/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75 g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P  < 0.05). Exercise improved clamp-derived insulin sensitivity by 75% (P  < 0.001) and decreased HOMA-IR by 15% (P  < 0.05). Training decreased plasma DPP-4 by 10% (421.8 ± 30.1 vs. 378.3 ± 32.5 ng/ml; P  < 0.04), and the decrease in DPP-4 was associated with clamp-derived insulin sensitivity (r  = −0.59; P  < 0.04), HOMA-IR (r  = 0.59; P  < 0.04) and fat oxidation (r  = −0.54; P  < 0.05). Increased fat oxidation also correlated with lower 2-h glucose levels (r  = −0.64; P  < 0.02). Exercise training reduces plasma DPP-4, which may be linked to elevated insulin sensitivity and fat oxidation. Maintaining low plasma DPP-4 concentrations is a potential mechanism whereby exercise plus weight loss prevents/delays the onset of type 2 diabetes in adults with metabolic syndrome.
Keywords: Impaired glucose tolerance; Obesity; Cytokines; Diabetes;

IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis by Vincent H.-S. Chang; Diane H.-A. Yang; Hsin-Hung Lin; Gregory Pearce; Clarence A. Ryan; Yu-Chi Chen (148-156).
► A 16-amino-acid peptide, IbACP, was isolated from leaves of sweet potato. ► IbACP contained no post-translational modifications and caused an alkalinizing response with plant cell media. ► IbACP regulated cellular proliferation and demonstrated anti-cancer effects via apoptosis.A 16-amino-acid peptide was isolated from the leaves of sweet potato. The peptide caused a rapid alkalinization response in tomato suspension culture media, a characteristic of defense peptides in plants. No post-translational modification was observed on the peptide according to MALDI-MS analysis. We have named the peptide Ipomoea batatas anti-cancer peptide (IbACP). IbACP also was shown with the ability to dose-dependently inhibit Panc-1, a pancreatic cancer line, cell proliferation. The morphological observations of the Panc-1 cells by phase contrast microscopy showed significant changes after treatment with IbACP. Moreover, caspase-3 and PARP [poly(ADP-ribose) polymerase] were activated by IbACP treatment, followed by cell death. An increase in the levels of cleaved caspase-3 and -9 was also detected by an immunoblot assay after treatment with IbACP. In addition, genomic DNA fragmentation and decreased cellular proliferation were induced when IbACP was supplied to the Panc-1 cells, further demonstrating its biological relevance. The combined data indicates that IbACP peptide may have an important role in the regulation of cellular proliferation by inducing and promoting apoptosis through the mitochondrial apoptotic pathway. This report also showed that IbACP peptide contains potent anti-cancer effects and may play an important role in herbal medicine development.
Keywords: Sweet potato; Anti-cancer peptide; Alkalinization assay; Apoptosis;