Peptides (v.64, #C)

Hemokinin-1 is an important mediator of endotoxin-induced acute airway inflammation in the mouse by Zsófia Hajna; Éva Borbély; Ágnes Kemény; Bálint Botz; László Kereskai; János Szolcsányi; Erika Pintér; Christopher J. Paige; Alexandra Berger; Zsuzsanna Helyes (1-7).
Hemokinin-1, the newest tachykinin encoded by the preprotachykinin C (Tac4) gene, is predominatly produced by immune cells. Similarly to substance P, it has the greatest affinity to the tachykinin NK1 receptor, but has different binding site and signaling mechanisms. Furthermore, several recent data indicate the existence of a not yet identified own receptor and divergent non-NK1-mediated actions. Since there is no information on its functions in the airways, we investigated its role in endotoxin-induced pulmonary inflammation.Acute pneumonitis was induced in Tac4 gene-deleted (Tac4−/−) mice compared to C57Bl/6 wildtypes by intranasal E. coli lipopolysaccharide (LPS). Airway responsiveness to inhaled carbachol was measured with unrestrained whole body plethysmography 24 h later. Semiquantitative histopathological scoring was performed; reactive oxygen species (ROS) production was measured with luminol bioluminescence, myeloperoxidase activity with spectrophotometry, and inflammatory cytokines with Luminex.All inflammatory parameters, such as histopathological alterations (perivascular edema, neutrophil/macrophage accumulation, goblet cell hyperplasia), myeloperoxidase activity, ROS production, as well as interleukin-1beta, interleukin-6, tumor necrosis factor alpha, monocyte chemoattractant protein-1 and keratinocyte chemoattractant concentrations were significantly diminished in the lung of Tac4−/− mice. However, bronchial hyperreactivity similarly developed in both groups. Interestingly, in LPS-treated Tac4−/− mouse lungs, bronchus-associated, large, follicle-like lymphoid structures developed.We provide the first evidence that hemokinin-1 plays a crucial pro-inflammatory role in the lung by increasing inflammatory cell activities, and might also be a specific regulator of lymphocyte functions.
Keywords: Acute pneumonitis; Tachykinin; Luminescence imaging; Reactive oxygen species;

Neuronostatin: Peripheral site of action in mouse stomach by Antonella Amato; Sara Baldassano; Gaetano Caldara; Flavia Mulè (8-13).
Neuronostatin is a 13-amino acid peptide encoded by somatostatin gene. It is distributed in different organs including gastrointestinal tract and has been involved in the control of food intake and gastrointestinal motility, likely through an action in the brain. So far, there are no reports about the occurrence of peripheral action sites in the gut. Therefore, the purpose of the present study was to examine, in the mouse, the effects of peripheral administration of neuronostatin on food intake within 24 h and on gastrointestinal motility and to analyse neuronostatin actions on the gastric and intestinal mechanical activity in isolated preparations in vitro. When compared with PBS-treated mice, intraperitoneal neuronostatin reduced food intake in doses ranging from 1 to 15 ng/g b.w. only in the first hour postinjection with a maximum effect obtained at the dose of 15 ng/g b.w. (−46.9%). The peptide (15 ng/g b.w.) significantly reduced gastric emptying rate (−31.1%) and gastrointestinal intestinal transit. Non-amidated neuronostatin failed to affect food intake, gastric emptying and intestinal transit, suggesting the specificity of action. In vitro, neuronostatin induced concentration-dependent gastric relaxation, which was abolished by tetrodotoxin. Neuronostatin failed to affect the spontaneous mechanical activity or the evoked cholinergic contractions in duodenum. These results suggest that exogenous neuronostatin is able to reduce mouse gastric motility by acting peripherally in the stomach, through intramural nervous plexuses. This indirectly action could cause reduction of food intake in the short term.
Keywords: Neuronostatin; Food intake; Gastric emptying; Intestinal transit;

The main objective of the study has been to show whether carnosine has positive effects on liver and lung tissues of rats exposed to a range of formaldehyde concentrations, and to explore how irisin expression and antioxidant capacity are altered in these tissues by carnosine supplementation. Sprague-Dawley type male rats were divided into 8 groups with 6 animals in each: (I) Control; no chemical supplementation); (II) sham (100 mg/kg/day carnosine); (III) low dose formaldehyde (LDFA) for 5 days/week; (IV) LDFA for 5 days/week and carnosine); (V) moderate dose formaldehyde (MDFA) for 5 days/week); (VI) MDFA for 5 days/week and carnosine; (VII) high dose formaldehyde (HDFA) for 5 days/week; (VIII) and HDFA for 5 days/week and carnosine. Sham and control groups were exposed to normal air. Irisin levels of the serum, liver and lung tissue supernatants were analyzed by ELISA, while the REL method was used to determine total oxidant/antioxidant capacity. Irisin production by the tissues was detected immunohistochemically. Increasing doses of FA decreased serum/tissue irisin and total antioxidant levels relative to the controls, as also to increases in TUNEL expressions, total oxidant level, oxidant and apoptosis index. Irisin expression was detected in hepatocyte and sinusoidal cells of the liver and parenchymal cells of the lung. In conclusion, while FA exposure reduces irisin and total oxidant in the serum, liver and lung tissues in a dose-dependent manner and increases the total antioxidant capacity, carnosine supplementation reduces the oxidative stress and restores the histopathological and biochemical signs.
Keywords: Formaldehyde; Carnosine; Irisin; Liver; Lungs; TAS/TOS;

Biomarkers offer new perspectives for a more personalized management of patients with heart failure (HF). Hyperparathyroidism is common in HF patients and parathyroid hormone (PTH) testing might provide added value for the prognostication of HF patients. Our objectives were therefore to determine the efficiency of multiple biomarker strategy based on PTH and natriuretic peptides measurement for the risk stratification of patients with HF. Circulating concentrations of bioactive PTH 1-84 and natriuretic peptides, B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP), were measured with automated immunoassays in 45 healthy individuals and 137 HF patients with reduced left ventricular ejection fraction. Circulating levels of PTH 1-84 and natriuretic peptides were significantly increased in HF patients in comparison to HF patients. Over a long-term follow-up, baseline PTH 1-84 levels were related to the risk of cardiovascular death. Furthermore, in multiple biomarker approach, PTH measurement was additive to BNP and NT-proBNP testing for the cardiovascular risk assessment of HF patients. In conclusion, the combination of PTH 1-84 and natriuretic peptides testing improves the prognostication of HF patients and might allowed more personalized approach for risk stratification and treatment selection in HF patients.
Keywords: PTH; NT-proBNP; BNP; Heart failure; Aldosterone; Prognosis;

Significance of serum neurokinin B and kisspeptin levels in the differential diagnosis of premature thelarche and idiopathic central precocious puberty by Ayhan Abacı; Gönül Çatlı; Ahmet Anık; Tuncay Küme; Özlem Gürsoy Çalan; Bumin Nuri Dündar; Ece Böber (29-33).
The aim of the present study was to investigate the diagnostic role of serum neurokinin B level and its relationship with kisspeptin and leptin, which are known to be involved in the initiation of pubertal process. Girls who presented with breast development (<8 years) were included in the study. All patients underwent bone age assessment. Basal levels of serum follicle stimulating hormone and luteinizing hormone were measured and gonadotropin releasing hormone stimulation test was performed. Patients with a bone age/chronological age ratio >1 and a peak luteinizing hormone response in gonadotropin releasing hormone stimulation test >5 mIU/L were included in the central precocious puberty group, while patients who did not meet these criteria were included in the premature thelarche group. Patients with organic pathologies were excluded. Healthy prepubertal girls with similar age were included as the control group. Leptin, kisspeptin and neurokinin B levels were measured by ELISA method. The study included 20 girls with idiopathic central precocious puberty 22 girls with premature thelarche and 24 prepubertal controls. While serum kisspeptin, leptin and neurokinin B levels were significantly higher in central precocious puberty and premature thelarche groups compared to controls, no significant difference was found between central precocious puberty and premature thelarche groups. Increased serum levels of leptin, kisspeptin and neurokinin B in patients with premature thelarche and central precocious puberty suggest that they take part during the initiation of pubertal process, however, these markers are not able to differentiate patients with central precocious puberty from premature thelarche.
Keywords: Precocious puberty; Premature thelarche; Leptin; Kisspeptin; Neurokinin B;

Antisense versus proopiomelanocortin mRNA reduces vascular risk in a murine model of type-2 diabetes following stress exposure in early post-natal life by Alberto Loizzo; Santi M. Spampinato; Andrea Fortuna; Stefano Vella; Fulvia Fabi; Paola Del Basso; Gabriele Campana; Stefano Loizzo (34-39).
Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides β-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l-NG-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.
Keywords: Antisense oligodeoxinucleotide; Aorta; Nitric oxide; β-Endorphin; Postnatal stress; Diabetes vascular complications;

Crosstalk between the microbiome and cancer cells by quorum sensing peptides by Evelien Wynendaele; Frederick Verbeke; Matthias D’Hondt; An Hendrix; Christophe Van De Wiele; Christian Burvenich; Kathelijne Peremans; Olivier De Wever; Marc Bracke; Bart De Spiegeleer (40-48).
To date, the precise role of the human microbiome in health and disease states remains largely undefined. Complex and selective crosstalk systems between the microbiome and mammalian cells are also not yet reported. Research up till now mainly focused on bacterial synthesis of virulence factors, reactive oxygen/nitrogen species (ROS/RNS) and hydrogen sulphide, as well as on the activation of exogenous mutagen precursors by intestinal bacteria. We discovered that certain quorum sensing peptides, produced by bacteria, interact with mammalian cells, in casu cancer cells: Phr0662 (Bacillus sp.), EntF-metabolite (Enterococcus faecium) and EDF-derived (Escherichia coli) peptides initiate HCT-8/E11 colon cancer cell invasion, with Phr0662 also promoting angiogenesis. Our findings thus indicate that the human microbiome, through their quorum sensing peptides, may be one of the factors responsible for cancer metastasis.
Keywords: Quorum sensing peptides; Colon cancer; Metastasis; Microbiome; Angiogenesis;

The aim of this study is to determine the antidiabetic activity of Ang-(1-7), an important component of the renin–angiotensin system, in a rat model of streptozotocin (STZ)-induced type 2 diabetes mellitus (DM). A total of 36 male Wistar rats were randomly divided into 3 groups: control group fed standard laboratory diet, DM group fed high-fat diet and injected with STZ, and Ang-(1-7) group receiving injection of STZ followed by Ang-(1-7) treatment. Body weight, blood glucose levels, fasting serum Ang II and insulin levels, and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. The pancreas was collected for histological examination and gene expression analysis. Notably, the Ang-(1-7) group showed a significant decrease in fasting blood glucose and serum Ang II levels and HOMA-IR values and increase in fasting serum insulin levels. Pancreatic β cells in the control and Ang-(1-7) groups were normally distributed in the center of pancreatic islets with large clear nuclei. In contrast, pancreatic β cells in the DM group had a marked shrinkage of the cytoplasm and condensation of nuclear chromatin. Ang-(1-7) treatment significantly facilitated insulin production by β cells in diabetic rats. The DM-associated elevation of inducible nitric oxide synthase (iNOS), caspase-3, caspase-9, caspase-8, and Bax and reduction of Bcl-2 was significantly reversed by Ang-(1-7) treatment. Taken together, Ang-(1-7) protects against STZ-induced DM through improvement of insulin resistance, insulin secretion, and pancreatic β cell survival, which is associated with reduction of iNOS expression and alteration of the Bcl-2 family.
Keywords: Angiotensin (1-7); Type 2 diabetes mellitus; Islet function; Oxidative stress; Cell apoptosis;

ML-18 is a non-peptide bombesin receptor subtype-3 antagonist which inhibits lung cancer growth by Terry W. Moody; Samuel A. Mantey; Paola Moreno; Taichi Nakamura; Enza Lacivita; Marcello Leopoldo; Robert T. Jensen (55-61).
Bombesin receptor subtype (BRS)-3 is a G protein coupled receptor (GPCR) for the bombesin (BB)-family of peptides. BRS-3 is an orphan GPCR and little is known of its physiological role due to the lack of specific agonists and antagonists. PD168368 is a nonpeptide antagonist for the neuromedin B (NMB) receptor (R) whereas PD176252 is a nonpeptide antagonist for the gastrin releasing peptide (GRP) R and NMBR but not BRS-3. Here nonpeptide analogs of PD176252 e.g. the S-enantiomer ML-18, and the R-enantiomer, EMY-98, were investigated as BRS-3 antagonists using lung cancer cells. ML-18 and EMY-98 inhibited specific 125I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to NCI-H1299 lung cancer cells stably transfected with BRS-3 with IC50 values of 4.8 and >100 μM, respectively. In contrast, ML-18 bound with lower affinity to the GRPR and NMBR with IC50 values of 16 and >100 μM, respectively. ML-18 (16 μM), but not its enantiomer EMY-98, inhibited the ability of 10 nM BA1 to elevate cytosolic Ca2+ in a reversible manner using lung cancer cells loaded with FURA2-AM. ML-18 (16 μM), but not EMY-98, inhibited the ability of 100 nM BA1 to cause tyrosine phosphorylation of the EGFR and ERK in lung cancer cells. ML-18 but not EMY-98 inhibited the proliferation of lung cancer cells. The results indicate that ML-18 is a nonpeptide BRS-3 antagonist that should serve as a template to improve potency and selectivity.
Keywords: Bombesin receptor subtype 3; Nonpeptide antagonist; Lung cancer; Proliferation;

Effects of centrally injected glucagon-like peptide-2 on gastric mucosal blood flow in rats: Possible mechanisms by Guldal Gulec Suyen; Naciye Isbil-Buyukcoskun; Betul Cam; Kasim Ozluk (62-66).
“Glucagon-like peptide-2” (GLP-2) is a peptide that is released from the enteroendocrine L cells in response to food in the gastrointestinal tract. Peripheral injection of GLP-2 has been shown to increase gastrointestinal blood flow, but effects of central GLP-2 on any vascular bed has not been studied yet. The aim of this study is to investigate the effects of various doses of intracerebroventricularly (i.c.v.)-injected GLP-2 on gastric mucosal blood flow (GMBF) and contribution of calcitonin gene related peptide (CGRP), nitric oxide synthase-nitric oxide (NOS-NO) and cyclooxygenase-prostaglandin (COX-PG) systems to the possible effect. The gastric chamber technique was used to determine GMBF. Urethane anesthesia was used throughout the recording procedure. Male Wistar rats were treated with GLP-2 (100, 150 ve 200 ng/10 μl; i.c.v.) or saline (10 μl; i.c.v.) in order to find out the effective dose of i.c.v. GLP-2 on GMBF. Then, CGRP receptor antagonist CGRP-(8-37) (10 μg/kg; s.c.), NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg; s.c.) or COX inhibitor indomethacin (5 mg/kg; i.p.) was injected before the effective dose of i.c.v. GLP-2. GMBF was measured continuously for 35 min following GLP-2 and recorded every fifth minute. Non-parametric Kruskal–Wallis test was used for statistical analysis. Differences were considered to be significant at p  < 0.05. GMBF increased rapidly following 100 ng GLP-2 injection and did not fall to the basal levels during 35 min. Other doses of i.c.v. GLP-2 did not produce any significant difference in GMBF. CGRP receptor antagonist, CGRP-(8-37) (10 μg/kg; s.c.) and COX inhibitor indomethacin (5 mg/kg; i.p.) significantly prevented the increase in GMBF due to GLP-2 (100 ng; i.c.v.), while l-NAME (30 mg/kg; s.c.) was ineffective. None of the drugs produced a significant change in GMBF when administered alone. Thus we suggest that, i.c.v. GLP-2 increases GMBF and CGRP and endogenous prostaglandins but not NO, contribute to this effect.
Keywords: Glucagon like peptide-2; CGRP-(8-37); l-NAME; Indomethacin; Gastric mucosal blood flow;

Dipyridamole-induced C-type natriuretic peptide mRNA overexpression in a minipig model of pacing-induced left ventricular dysfunction by M. Cabiati; S. Burchielli; M. Matteucci; B. Svezia; L. Panchetti; C. Caselli; T. Prescimone; M.A. Morales; S. Del Ry (67-73).
Dipyridamole (DP) restores ischemic tissue blood flow stimulating angiogenesis in eNOS-dependent pathways. C-type natriuretic peptide (CNP) is expected to mimic the migration-stimulatory effect of NO via a cGMP-dependent mechanism. Aim of this study was to assess the role of concomitant treatment with DP on CNP levels in blood and myocardial tissue of minipigs with left ventricular dysfunction (LVD) induced by pacing at 200 bpm in the right ventricular apex. Minipigs with DP therapy (DP+, n  = 4) or placebo (DP−, n  = 4) and controls (C-SHAM, n=4) underwent 2D-EchoDoppler examination and blood collection before and after 4 weeks of pacing, when cardiac tissue was collected. Histological/immunohistochemical analyses were performed. CNP levels were determined by radioimmunoassay; cardiac CNP, BNP, natriuretic receptors expression by Real-Time PCR. After pacing, cardiac parameters resulted less impaired in DP+ compared to DP−. Histological sections presented normal morphology while the arteriolar density resulted: C-SHAM: 9.0 ± 1.2; DP−: 4.9 ± 0.3; DP+: 6.5 ± 0.6 number/mm2; C-SHAM vs DP− and DP+ p  = 0.004, p  = 0.04, respectively. CNP mRNA resulted lower in DP− compared to C-SHAM and DP+ as well as NPR-B (p  = 0.011, DP− vs DP+). Both NPR-A/NPR-C mRNA expressions were significantly (p  < 0.001) lower both in DP− and DP+ compared to C-SHAM. BNP mRNA was higher in LVD. CNP plasma levels showed a similar trend with respect to gene expression (C-SHAM: 30.5 ± 15; DP−: 18.6 ± 5.5; DP+: 21.2 ± 4.7 pg/ml). These data suggest that DP may serve as a preconditioning agent to increase the protective CNP-mediated endocrine response in LVD. This response, mediated by its specific receptor NPR-B, may offer new insights into molecular targets for treatment of LVD.
Keywords: C-type natriuretic peptide; Dipyridamole; mRNA; Left ventricular dysfunction; RIA;

The effects of exercise training programs on plasma concentrations of proenkephalin Peptide F and catecholamines by William J. Kraemer; Scott E. Gordon; Maren S. Fragala; Jill A. Bush; Tunde K. Szivak; Shawn D. Flanagan; David R. Hooper; David P. Looney; N. Travis Triplett; William H. DuPont; Joseph E. Dziados; Louis J. Marchitelli; John F. Patton (74-81).
To determine if exercise training alters the pattern and magnitude of plasma concentrations of proenkephalin Peptide F and epinephrine, plasma proenkephalin [107–140] Peptide Fir and catecholamines were examined pre-training (T-1), and after 4- (T-2), 8- (T-3), and 12-weeks (T-4) of training. 26 healthy men were matched and randomly assigned to one of three groups: heavy resistance strength training (Strength, n  = 9), high intensity endurance training (Endurance, n  = 8), or both training modalities combined (Combined, n  = 9). Blood was collected using a syringe with a cannula inserted into a superficial arm vein with samples collected at rest, after each 7 min stage and 5 and 15 min into recovery. With training, all groups observed shifted plasma Peptide F responses to graded exercise, where significant increases were observed at lower exercise intensities. Increases in plasma epinephrine with exercise were observed in all groups. The Combined group saw increases at 25% at T-3 and for 50% at T-2, T-3, and T-4 which was higher than T-1. The Endurance group demonstrated increases for 50% at T-1, T-2, T-3 but not at T-4. The plasma epinephrine response to graded exercise was reduced in the Strength group. Increases in plasma norepinephrine above rest were observed starting at 50% ▪. The Strength group demonstrated a significant reduction in norepinephrine observed at 100% at T-3 and T-4. Peptide F and catecholamines responses to graded exercise can be altered by different types of physical exercise training. Simultaneous high intensity training may produce adrenal medulla exhaustion when compared to single mode training.
Keywords: Proenkephalins; Opioid peptides; Epinephrine; Norepinephrine; Resistance training; Endurance training;