Peptides (v.55, #C)

Uveal melanoma expresses NK-1 receptors and cyclosporin A induces apoptosis in human melanoma cell lines overexpressing the NK-1 receptor by Ana González-Ortega; Elia Sánchez-Vaderrábanos; Susana Ramiro-Fuentes; Manuel Vicente Salinas-Martín; Andrés Carranza; Rafael Coveñas; Miguel Muñoz (1-12).
Substance P and neurokinin-1 (NK-1) receptor antagonists respectively induce proliferation and growth inhibition in human melanoma cell lines. The presence of NK-1 receptors in human melanoma cell lines and samples has been reported, but the presence of NK-1 receptors has not been demonstrated in uveal melanomas. It is known that melanoma express the tachykinin 1 receptor (TAC1R) gene. This gene is overexpressed in several human cancer cell lines, but such overexpression is currently unknown in human malignant melanoma cell lines (COLO 858, MEL HO, COLO 679). In this study, we attempt to demonstrate the overexpression of the TAC1R gene in such cells. We performed an in vitro study by real-time quantitative RT-PCR for TAC1R and found that the NK-1 receptor was overexpressed in the three human melanoma cell lines studied. Using a knockdown method, we demonstrate that the NK-1 receptor is involved in the viability of the COLO 858 melanoma cell line. Immunohistochemistry was also used to demonstrate NK-1 receptors in uveal melanoma samples. We observed that NK-1 receptors were present in the 21/21 uveal melanomas. In addition, cyclosporin A inhibited the growth of the three melanoma cell lines studied in a dose-dependent manner, and after the administration of this immunosuppresive drug apoptosis was observed. This indicates at least that the antitumor action of cyclosporin A is mediated by the NK-1 receptor. Our findings suggest that the NK-1 receptor could be a promising target in the treatment of human melanomas.
Keywords: Apoptosis; COLO 679; COLO 858; Cyclosporin A; MEL HO; NK-1 receptor; Substance P; TAC1R; Uveal melanoma;

Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease by Cabiati Manuela; Sabatino Laura; Svezia Benedetta; Caruso Raffaele; Verde Alessandro; Caselli Chiara; Prescimone Tommaso; Giannessi Daniela; D.R. Silvia (13-16).
Adrenomedullin (ADM) is a vasodilatory peptide expressed in many tissues. Its levels are elevated in various diseases including chronic heart failure (CHF) and it has been suggested that the up-regulation of ADM in cardiac disease represents a protective mechanism. Similarly, intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is considered a potential endogenous protector of the heart. Previous studies demonstrated that in CHF patients, elevated plasma concentrations of ADM and IMD reflect the patient's disease severity and prognosis, while the behavior of mRNA expression is not known. The aim of this study was to evaluate ADM/IDM transcriptomic profiling in human leukocytes of CHF patients as a function of clinical severity, assessing 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 from CHF patients (NYHA I–II n  = 10; NYHA III–IV n  = 14) with PAXgene Blood RNA Kit. Significantly higher levels of ADM and IMD mRNA were found in CHF as a function of clinical severity (ADM: C  = 0.03 ± 0.013, NYHA I–II = 0.11 ± 0.084, NYHA III–IV = 11.46 ± 4.72, p  = 0.037 C vs NYHA III–IV, p  = 0.028 NYHA I–II vs NYHA III–IV; IMD: C  = 0.158 ± 0.041, NYHA I–II = 0.93 ± 0.40, NYHA III–IV = 2.6 ± 0.67, p  = 0.014 C vs NYHA III–IV, p  = 0.014 NYHA I–II vs NYHA III–IV). This study highlights, for the first time, the possibility of evaluating ADM and IMD mRNA expression in human whole blood samples by Real-Time PCR study providing further relevant information and providing a more complete interpretation of the pathophysiology of the disease.
Keywords: Adrenomedullin; Intermedin; Real-Time PCR; Leukocyte mRNA expression; Human leukocytes; Heart failure;

In vitro activity and in vivo animal model efficacy of IB-367 alone and in combination with imipenem and colistin against Gram-negative bacteria by Oriana Simonetti; Oscar Cirioni; Roberto Ghiselli; Fiorenza Orlando; Carmela Silvestri; Susanna Mazzocato; Wojciech Kamysz; Elzbieta Kamysz; Mauro Provinciali; Andrea Giacometti; Mario Guerrieri; Annamaria Offidani (17-22).
The aim of our study was to evaluate the in vitro activity of IB-367 and its bactericidal effect for Pseudomonas aeruginosa and Escherichia coli, associated to a synergic study to test the antibiotic combinations between the peptide and colistin or imipenem. Minimum inhibitory concentrations (MICs), the minimum bactericidal concentrations (MBCs), the synergy test and killing study were carried out to evaluate the IB-367 activity. In the in vivo model, a wound was incised through the panniculus carnosus of BALB/c mice, and then inoculated with 5 × 107 colony-forming units of P. aeruginosa and E. coli. For each strain, the study included an infected or not infected group that did not receive any treatment, and five contaminated groups treated with local IB- 367, intraperitoneal imipenem, intraperitoneal colistin, topical IB-367 local plus intraperitoneal imipenem or intraperitoneal colistin. All isolates were inhibited by IB-367 at concentrations of 4–64 mg/l. Killing by IB-367 was shown to be very rapid: its activity on all Gram-negative bacteria was completed within a 40 min exposure period at a concentration of 2× MIC/l. Synergy was demonstrated when IB-367 was combined with colistin or imipenem. In in vivo studies, the groups treated with topical IB-367 and intraperitoneal colistin showed the best results in terms of bacterial load inhibition either for Pseudomonas or for E. coli. The good in vitro activity and in vivo efficacy, as well as, the synergic interactions with antibiotics suggest that IB-367 is a promising candidate for potential application in the treatment of wound Gram-negative infections.
Keywords: IB-367; Wound infection; Animal model; Pseudomonas aeruginosa; Escherichia coli;

Insulin-releasing and cytotoxic properties of the frog skin peptide, tigerinin-1R: a structure–activity study by Dinesh Srinivasan; Opeolu O. Ojo; Yasser H.A. Abdel-Wahab; Peter R. Flatt; Laure Guilhaudis; J. Michael Conlon (23-31).
The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH2) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P  < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P  < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC50  = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P  < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.
Keywords: Frog skin peptide; Tigerinin; Incretin; Cytotoxicity, Type 2 diabetes mellitus;

The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt = 2′,6′-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC–MS) and tandem mass spectrometry (LC–MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.
Keywords: Opioid peptides; Solid-phase peptide synthesis; Cyclization in solution; On-resin cyclization; Liquid chromatography combined with on-line mass spectrometry (LC–MS); Tandem mass spectrometry (MS/MS);

The site(s) of action that control the reduction of food intake in response to the amphibian skin peptide bombesin (Bn) has been determined to be the area supplied by the celiac artery (CA), i.e., the stomach and the upper duodenum. Here, we investigated the gastrointestinal site(s) of action which controls meal size (MS) (normal rat chow) and intermeal interval length (IMI) by the mammalian homologues of Bn gastrin releasing peptides (GRP-10, GRP-27 and GRP-29, 0.01, 0.05, 0.1, 0.2 and 0.5 nmol/kg) infused in the CA, the cranial mesenteric artery (CMA, supplying the small and large intestine), the femoral artery (FA, control) and the portal vein (PV, draining the gastrointestinal tract, control) in freely fed rats immediately prior to the onset of the dark cycle. We found that (1) GRP-29 (0.05, 0.1, 0.2 and 0.5 nmol/kg) and GRP-27 (0.2 and 0.5 nmol/kg) in the CA and GRP-29 (0.5 nmol/kg) in the CMA reduced the MS relative to saline, (2) GRP-29 (0.1, 0.2 and 0.5 nmol/kg) and GRP-27 (0.2 and 0.5 nmol/kg) in the CA prolonged the IMI, (3) GRP-29 (0.1, 0.2 and 0.5 nmol/kg) in the CA and GRP-29 (0.5 nmol/kg) in the CMA increased the satiety ratio (SR, IMI/MS – the amount of food consumed per a given unit of time) and (4) neither peptide nor route showed any effect on the second MS. These results support an upper gastrointestinal site of action for MS and IMI length by GRP-27 and GRP-29, which is most likely the stomach and/or the duodenum.
Keywords: GRP; Celiac artery; Cranial mesenteric artery; Portal vein; Food intake;

Opiorphin increases blood pressure of conscious rats through renin-angiotensin system (RAS) by Yuan Fang; Shuo Li; Huabin Zhou; Xiaozhu Tian; Shuangyu Lv; Qiang Chen (47-51).
Human opiorphin is a recently identified endogenous pentapeptide, encoded by ProL1 multigenes family that contributes to cardiovascular modulation. The aim of this study was to evaluate the effect of opiorphin through intravenous injection (i.v.) on mean arterial pressure (MAP) regulation. To investigate the bioactivity of opiorphin, a rat cannulation model was developed for MAP measurement and blood sampling. In our present study, opiorphin (200–700 nmol/kg) increased MAP in dose-related and time-dependent manner in conscious rats, which associated highly with the elevation of angiotensin II (AngII) levels in serum. Furthermore, the MAP elevation induced by opiorphin was completely blocked by AngII receptor antagonist valsartan and partially attenuated by angiotensin-converting enzyme inhibitor captopril. Finally, we tested the effect of opiorphin in hypoxia condition, which exhibited that opiorphin reversed hypoxia induced hypotension in conscious rats. Taken together, these results indicated that opiorphin may play an important role in the modulation of blood pressure through AngII dependent pathway, which may help future development of potent clinical therapeutics for emergency treatment.
Keywords: Opiorphin; Blood pressure; Vasopressor; AngII; Hypoxia;

GLP-1 amidation efficiency along the length of the intestine in mice, rats and pigs and in GLP-1 secreting cell lines by Rune Ehrenreich Kuhre; Nicolai Wewer Albrechtsen; Johanne Agerlin Windeløv; Berit Svendsen; Bolette Hartmann; Jens Juul Holst (52-57).
Measurements of plasma concentrations of the incretin hormone GLP-1 are complex because of extensive molecular heterogeneity. This is partly due to a varying and incompletely known degree of C-terminal amidation. Given that virtually all GLP-1 assays rely on a C-terminal antibody, it is essential to know whether or not the molecule one wants to measure is amidated. We performed a detailed analysis of extractable GLP-1 from duodenum, proximal jejunum, distal ileum, caecum, proximal colon and distal colon of mice (n  = 9), rats (n  = 9) and pigs (n  = 8) and determined the degree of amidation and whether this varied with the six different locations. We also analyzed the amidation in 3 GLP-1 secreting cell lines (GLUTag, NCI-H716 and STC-1). To our surprise there were marked differences between the 3 species with respect to the concentration of GLP-1 in gut. In the mouse, concentrations increased continuously along the intestine all the way to the rectum, but were highest in the distal ileum and proximal colon of the rat. In the pig, very little or no GLP-1 was present before the distal ileum with similar levels from ileum to distal colon. In the mouse, GLP-1 was extensively amidated at all sampling sites, whereas rats and pigs on average had around 2.5 and 4 times higher levels of amidated compared to non-amidated GLP-1, although the ratio varied depending upon the location. GLUTag, NCI-H716 and STC-1 cells all exhibited partial amidation with 2–4 times higher levels of amidated compared to non-amidated GLP-1.
Keywords: GLP-1; Peptide's assays; GLP-1 measurement;

Inflammation-induced functional connectivity of melanin-concentrating hormone and IL-10 by Dimitrios C. Ziogas; Apostolos K.A. Karagiannis; Brenda M. Geiger; Beatriz Gras-Miralles; Robert Najarian; Ofer Reizes; Leo R. Fitzpatrick; Efi Kokkotou (58-64).
Melanin-concentrating hormone (MCH) was identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, similarly to most of the brain peptides, MCH is also produced in the gastrointestinal system and can act locally as an immunomodulator. We have previously reported high expression of MCH and its receptor MCHR1 in the affected mucosa of patients with inflammatory bowel disease. Furthermore, MCH deficiency in mice attenuated experimental colitis, pointing to MCH as a mediator of intestinal inflammation. In the present study, in order to gain further insights into the underlying mechanisms of such effects of MCH, we treated mice with established experimental colitis due to IL-10 deficiency with a MCHR1 antagonist (DABA-822). While treatment with the same drug was successful in attenuating TNBS-induced colitis in previous studies, it offered no benefit to the IL-10 knockout mouse model, suggesting that perhaps IL-10 is a downstream target of MCH. Indeed, in experiments focusing on monocytes, we found that treatment with MCH inhibited LPS-mediated IL-10 upregulation. Conversely, in the same cells, exogenous IL-10 prevented LPS-induced MCHR1 expression. Taken together, these findings indicate a functional cross-talk between MCH and IL-10 which prevents resolution of inflammation.
Keywords: Melanin-concentrating hormone; IL-10; Monocytes; Cytokines; Intestinal inflammation; Neuropeptides;

The use of versatile plant antimicrobial peptides in agribusiness and human health by Elizabete de Souza Cândido; Marlon Henrique e Silva Cardoso; Daniel Amaro Sousa; Juliane Cançado Viana; Nelson Gomes de Oliveira-Júnior; Vívian Miranda; Octávio Luiz Franco (65-78).
Plant immune responses involve a wide diversity of physiological reactions that are induced by the recognition of pathogens, such as hypersensitive responses, cell wall modifications, and the synthesis of antimicrobial molecules including antimicrobial peptides (AMPs). These proteinaceous molecules have been widely studied, presenting peculiar characteristics such as conserved domains and a conserved disulfide bond pattern. Currently, many AMP classes with diverse modes of action are known, having been isolated from a large number of organisms. Plant AMPs comprise an interesting source of studies nowadays, and among these there are reports of different classes, including defensins, albumins, cyclotides, snakins and several others. These peptides have been widely used in works that pursue human disease control, including nosocomial infections, as well as for agricultural purposes. In this context, this review will focus on the relevance of the structural-function relations of AMPs derived from plants and their proper use in applications for human health and agribusiness.
Keywords: Plant defense; Antimicrobial peptides; Biotechnological properties; Antibiotics;

Solubilization and reconstitution of the mu-opioid receptor expressed in human neuronal SH-SY5Y and CHO cells by Franck Talmont; Lionel Moulédous; Catherine Mollereau; Jean-Marie Zajac (79-84).
The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.
Keywords: Neuropeptide; G protein-coupled receptor; GPCR; Solubilization; Reconstitution; Opioid receptors;

Irisin: A potentially candidate marker for myocardial infarction by Tuncay Kuloglu; Suna Aydin; Mehmet Nesimi Eren; Musa Yilmaz; İbrahim Sahin; Mehmet Kalayci; Emine Sarman; Nalan Kaya; Osman Fatih Yilmaz; Ahmet Turk; Yalcin Aydin; Mehmet Hanifi Yalcin; Nimet Uras; Ali Gurel; Selcuk İlhan; Evrim Gul; Suleyman Aydin (85-91).
Myocardial infarction (MI) causes energy depletion through imbalance between coronary blood supply and myocardial demand. Irisin produced by the heart reduces ATP production by increasing heat generation. Energy depletion affects irisin concentration in circulation and cardiac tissues, suggesting an association with MI. We examined: (1) irisin expression immunohistochemically in rat heart, skeletal muscle, kidney and liver in isoproterenol (ISO)-induced MI, and (2) serum irisin concentration by ELISA. Rats were randomly allocated into 6 groups (n  = 6), (i) control, (ii) ISO (1 h), (iii) ISO (2 h), (iv) ISO (4 h), (v) ISO (6 h), and (vi) ISO (24 h), 200 mg ISO in each case. Rats were decapitated and the blood and tissues collected for irisin analysis. Blood was centrifuged at 1792 g for 5 min. Tissues were washed with saline and fixed in 10% formalin for histology. Serum irisin levels gradually decreased from 1 h to 24 h in MI rats compared with controls, the minimum being at 2 h, increasing again after 6 h. Cardiac muscle cells, glomerular, peritubular renal cortical interstitial cells, hepatocytes and liver sinusoidal cells and perimysium, endomysium and nucleoi of skeletal muscle were irisin positive, but its synthesis decreased 1–4 h after MI. At all time-points, irisin increased near myocardial connective tissue, with production in skeletal muscle, liver and kidney recovering after 6 h, although slower than controls. Unique insight into the pathogenesis of MI is shown, and the gradually decrease of serum irisin might be a diagnostic marker for MI.
Keywords: Irisin; Myocardial infarction; Isoproterenol; Heart; Skeletal muscle; Kidneys;

Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and they can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both native and recombinant peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide.
Keywords: Plant glycine-rich proteins; Antimicrobial peptides; Ab initio molecular modelling; Molecular dynamics; Solvated potential energy;

It has been proposed that some neuropeptides may be anchored to the cell membranes prior to attaching to the adjacent active sites of transmembrane receptors. The three amphibian skin neuropeptides signiferin 1 [RLCIPYIIPC(OH)] (smooth muscle active and immunomodulator), riparin 1.1 [[RLCIPVIFPC(OH)] (immunomodulator) and rothein 1 [SVSNIPESIGF(OH)] (immunomodulator) act via CCK2 transmembrane receptors. A combination of 31P and 2H solid state NMR studies of each of these three peptides in eukaryotic phospholipid models at 25 °C shows that rothein 1 does not interact with the membrane at all. In contrast, both of the cyclic disulfides signiferin 1 and riparin 1.1 interact with phospholipid head groups and partially penetrate into the upper leaflet of the model bilayer, but to different extents. These interactions are not sufficiently effective to cause disruption of the lipid bilayer since the peptides are not antimicrobial, anticancer, antifungal nor active against enveloped viruses.
Keywords: Signiferin 1; Riparin 1.1; Rothein 1; 3D NMR structures; Smooth muscle activity; Immunological activity; Trans membrane CCK2 receptors; 31P and 2H solid state NMR; Binding to eukaryotic model phospholipids;

Ghrelin receptor agonist, GHRP-2, produces antinociceptive effects at the supraspinal level via the opioid receptor in mice by Ping Zeng; Shu Li; Yue-hui Zheng; Fu-Yan Liu; Jing-lei Wang; Da-lei Zhang; Jie Wei (103-109).
GHRP-2 is a synthetic agonist of ghrelin receptor. GHRP-2 has similar physiological functions with ghrelin. In our previous study, ghrelin (i.c.v.) could induce analgesic effect through an interaction with GHS-R1α and with the central opioid system in the acute pain in mice. To date, the function of GHRP-2 in pain processing was not understood. Therefore the aim of this study was to investigate the effects of GHRP-2 on pain modulation at supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of GHRP-2 (0.1, 0.3, 1, 3 and 10 nmol/L) produced a concentration- and time-related antinociceptive effect. This effect could be fully antagonized by GHS-R1α antagonist [d-Lys3]-GHRP-6, indicating that the analgesic effect induced by GHRP-2 is mediated through the activation of GHS-R1α. Interestingly, naloxone, naltrindole and nor-binaltorphimine, but not β-funaltrexamine, could also block the analgesic effect markedly, suggesting that δ- and κ-opioid receptor is involved in the analgesic response evoked by GHRP-2. Moreover, i.c.v. administration of GHRP-2 potentiated the analgesic effect induced by morphine (i.c.v., 1 nmol/L) and this potentiated effect could not be reversed by [d-Lys3]-GHRP-6. Thus these findings may be a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation. Furthermore, GHRP-2 may be a promising peptide for developing new analgesic drugs.
Keywords: GHRP-2; Growth hormone secretagogue receptor 1 alpha (GHS-R1a); Opioid receptor; Antinociception; Morphine; Tail withdrawal test;

Blockade of central delta-opioid receptors inhibits salt appetite in sodium-depleted rats by A.I.R. Nascimento; H.S. Ferreira; D.R. Cerqueira; J.B. Fregoneze (110-119).
Various studies have investigated the role of central opioid peptides in feeding behavior; however, only a few have addressed the participation of opioids in the control of salt appetite. The present study investigated the effect of intracerebroventricular injections of the δ-opioid antagonist, naltrindole (5, 10 and 20 nmol/rat) and the agonist, deltorphin II (2.5, 5, 10 and 20 nmol/rat) on salt intake. Two protocols for inducing salt intake were used: sodium-depletion and the central injection of angiotensin II. In addition, the effect of a central δ-opioid receptor blockade on locomotor activity, on palatable solution intake (0.1% saccharin) and on blood pressure was also studied. The blockade of central δ-opioid receptors inhibits salt intake in sodium-depleted rats, while the pharmacological stimulation of these receptors increases salt intake in sodium-replete animals. Furthermore, the blockade of central δ-opioid receptors inhibits salt intake induced by central angiotensinergic stimulation. These data suggest that during sodium-depletion activation of the δ-opioid receptors regulates salt appetite to correct the sodium imbalance and it is possible that an interaction between opioidergic and angiotensinergic brain system participates in this control. Under normonatremic conditions, δ-opioid receptors may be necessary to modulate sodium intake, a response that could be mediated by angiotensin II. The decrease in salt intake following central δ-opioid receptors blockade does not appear to be due to a general inhibition of locomotor activity, changes in palatability or in blood pressure.
Keywords: Salt intake; Delta-opioid receptors; Angiotensin II; Sodium-depletion;

CCK-58 prolongs the intermeal interval, whereas CCK-8 reduces this interval: Not all forms of cholecystokinin have equal bioactivity by Ayman I. Sayegh; Martha C. Washington; Shannon J. Raboin; Amnah H. Aglan; Joseph R. Reeve (120-125).
It has been accepted for decades that “all forms of cholecystokinin (CCK) have equal bioactivity,” despite accumulating evidence to the contrary. To challenge this concept, we compared two feeding responses, meal size (MS, 10% sucrose) and intermeal interval (IMI), in response to CCK-58, which is the major endocrine form of CCK, and CCK-8, which is the most abundantly utilized form. Doses (0, 0.1, 0.5, 0.75, 1, 3 and 5 nmol/kg) were administered intraperitoneally over a 210-min test to Sprague Dawley rats that had been food-deprived overnight. We found that (1) all doses of CCK-58, except the lowest dose, and all doses of CCK-8, except the lowest two doses, reduced food intake more than vehicle did; (2) at two doses, 0.75 and 3 nmol/kg, CCK-58 increased the IMI, while CCK-8 failed to alter this feeding response; and (3) CCK-58, at all but the lowest two doses, increased the satiety ratio (IMI between first and second meals (min) divided by first MS (ml)) relative to vehicle, while CCK-8 did not affect this value. These findings demonstrate that the only circulating form of CCK in rats, CCK-58, prolongs the IMI more than CCK-8, the peptide generally utilized in feeding studies. Taken together, these results add to a growing list of functions where CCK-8 and CCK-58 express qualitatively different bioactivities. In conclusion, the hypothesis that “all forms of cholecystokinin (CCK) have equal bioactivity” is not supported.
Keywords: Cholecystokinin; Satiation; Satiety; Satiety ratio; Intermeal interval;

Protective effect of oxytocin on ovarian ischemia-reperfusion injury in rats by Ali Akdemir; Oytun Erbas; Funda Gode; Mete Ergenoglu; Ozgur Yeniel; Fatih Oltulu; Altug Yavasoglu; Dilek Taskiran (126-130).
Oxytocin (OT), a neurohypophysial nonapeptide, plays dual role as a neurotransmitter/neuromodulator and a hormone. It has also well known protective properties against ischemia/reperfusion organ damage. This study investigated the effect of OT on experimentally induced ovarian torsion/de-torsion ischemia/reperfusion (I/R) injury in rats. Sprague-Dawley rats were assigned to five treatment groups (n  = 7/group): Group 1, sham-operated; Group 2, torsion; Group 3, 80 IU/kg of OT administration 30 min prior to torsion; Group 4, torsion/de-torsion; and Group 5, torsion followed by 80 IU/kg of OT administration 30 min prior to de-torsion. OT administration significantly decreased the tissue malondialdehyde (MDA) levels in both the torsion and OT group (Group 3), and torsion/de-torsion OT group (Group 5) in comparison with the torsion-only group (Group 2) and torsion/de-torsion group (Group 4). Histopathological finding scores including follicular degeneration, edema, hemorrhage, vascular congestion, and infiltration by inflammatory cells were found to be significantly decreased in the torsion and OT group (Group 3), and torsion/de-torsion OT group (Group 5) when compared with the torsion-only group (Group 2) and torsion/de-torsion group (Group 4). In conclusion, these results, verified with histopathologic evaluation and biochemical assays, suggest a probable protective role for OT in ischemia and I/R injury in rat ovaries.
Keywords: Oxytocin; Ischemia/reperfusion; Ovary; Torsion; Protection;

Usefulness of catestatin to predict malignant arrhythmia in patients with acute myocardial infarction by Zhiqiang Pei; Dengfeng Ma; Lei Ji; Jing Zhang; Jinsheng Su; Weizhen Xue; Xiaoping Chen; Weishu Wang (131-135).
Catestatin (CST) displays potent vasodilatory effect and acts on lowering blood pressure in vivo. The clinical utility of CST in patients with acute myocardial infarction (AMI) has not been clearly delineated. The aim of this study was to investigate the predictive value of CST for the development of in-hospital malignant arrhythmia and other adverse cardiac events in patients with AMI. A total of 125 consecutive patients diagnosed with AMI were included. The clinical characteristics and previous history of the patients were collected. Malignant arrhythmia and other major adverse cardiac events (MACE) such as postinfarction angina pectoris or reinfarction and death were recorded during hospitalization. The levels of plasma CST, norepinephrine (NE) and amino-terminal pro-brain sodium peptides (NT-proBNP) were determined by sandwich ELISA. A multiple logistic regression model was used to predict the influence factors of malignant arrhythmia and other MACE during hospitalization of AMI patients. The results showed that the levels of plasma cystatin-C (CysC), high sensitivity C-reactive protein (hs-CRP), NE and NT-proBNP increased in a CST concentration dependent manner. The incidence of malignant arrhythmia significantly increased as the elevation of CST level (P  < 0.05). Age, CST and NT-proBNP were independent predictors for the MACE occurred during hospitalization. Increased blood glucose (≥6.1 mmol/L) and CST were independent predictors for the complicated malignant arrhythmia of AMI patients. These data demonstrated that CST can be used as a new biological marker for prediction of malignant arrhythmia in patients with AMI.
Keywords: Acute myocardial infarction; Catestatin; Adverse cardiac events;

Preventive effect of rikkunshito on gastric motor function inhibited by l-dopa in rats by Lixin Wang; Sachiko Mogami; Hiroshi Karasawa; Chihiro Yamada; Seiichi Yakabi; Koji Yakabi; Tomohisa Hattori; Yvette Taché (136-144).
We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinson's disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mg kg−1) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg−1) reduced the LD/CD (20/2 mg kg−1) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys3]-GHRP-6 (1 mg kg−1) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg−1) blocked LD/CD (20/2 mg kg−1)-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinson's disease rat model, rikkunshito (1.0 g kg−1, og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms.
Keywords: Gastric motility; Ghrelin; l-dopa; Parkinson's disease; Rats; Rikkunshito;

Pharmacological characterization of endomorphin-2-based cyclic pentapeptides with methylated phenylalanine residues by Renata Perlikowska; Davide Malfacini; Maria Camilla Cerlesi; Girolamo Calo’; Justyna Piekielna; Léonore Floriot; Tiphaine Henry; Jean Claude do-Rego; Csaba Tömböly; Alicja Kluczyk; Anna Janecka (145-150).
As part of our continuing studies on the structure–activity relationships of cyclic pentapeptides based on the structure of endomorphin-2, we report here the synthesis and biological activities of a new series of analogs incorporating 2′, 3′ or 4′-methylphenylalanine (MePhe) residues into positions 3 or 4 of the parent cyclopeptide, Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (Dmt = 2′,6′-dimethyltyrosine). Analogs with MePhe in position 4 showed a row of magnitude increased μ-opioid receptor (MOP receptor) affinity as compared with a parent compound. The in vitro potencies of the new analogs were determined in calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. All analogs were strong μ/κ (MOP/KOP) receptor agonists and weak δ (DOP) receptor agonists. In the in vivo hot-plate test in mice, the MePhe4-modified peptides showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration which was most likely due to the concomitant activation of more than one opioid receptor type.
Keywords: Solid phase peptide synthesis; Cyclic peptides; Opioid receptors; Receptor binding; Calcium mobilization; Hot-plate test;

Circulating levels of the vasoactive peptide urotensin II in patients with acute coronary syndrome and stable coronary artery disease by Hamood Al Kindi; Anouar Hafiane; Zhipeng You; Isabella Albanese; Louise Pilote; Jacques Genest; Adel Schwertani (151-157).
Urotensin II (UII) is a vasoactive peptide with various roles in cardiovascular physiology and pathophysiology. There is an accumulating evidence implicating UII in atherosclerosis and coronary artery disease, making it an important target in acute coronary syndrome (ACS). In this study, we sought to determine the plasma levels of UII in ACS patients within 48 h of clinical presentation and after a 12-week recovery period. We compared them to patients with stable coronary artery disease (CAD) and a control group of normolipidemic subjects without known CAD. Using a highly sensitive ELISA technique, we measured plasma UII in 27 ACS patients, 26 stable CAD patients and 22 age-matched controls. ACS patients had significantly elevated plasma UII during the first 48 h of clinical presentation compared to stable CAD patients and controls. We also found significant positive correlations between UII and CRP and with triglycerides and a significant negative correlation between UII and EF. There was no correlation with LDL-C. In conclusion, plasma UII levels were elevated in patients with acute coronary syndrome, particularly immediately after clinical presentation. This suggests an upregulation of UII expression in ACS.
Keywords: Acute coronary syndrome; Coronary artery disease; Plasma; Urotensin II; UII; Urotensin II-related peptide; URP; UT; Troponin; CRP; Triglycerides; Ejection fraction;

Cross talk between angiotensin-(1–7)/Mas axis and sirtuins in adipose tissue and metabolism of high-fat feed mice by João Marcus Oliveira Andrade; Alanna Fernandes Paraíso; Zélia Menezes Garcia; Adaliene Versiani Matos Ferreira; Ruben D.M. Sinisterra; Frederico B. Sousa; André Luiz Sena Guimarães; Alfredo Maurício Batista de Paula; Maria José Campagnole-Santos; Robson Augusto dos Santos; Sérgio Henrique Sousa Santos (158-165).
Angiotensin-(1–7) and resveratrol have been described as new potential therapeutic tools on treating and preventing metabolic disorders. In the present study we aimed to evaluate the effect of an oral formulation of angiotensin-(1–7) [Ang-(1–7)] included in HPB-cyclodextrin and resveratrol (RSV), in modulation of sirtuin and renin-angiotensin system (RAS) in adipose tissue of mice treated with a high-fat diet (HFD). We observed that HFD + Ang-(1–7) and HFD + RSV groups presented marked decrease in the adipose tissue mass. Furthermore, these animals showed improved insulin-sensitivity and glucose tolerance as well as lower plasma levels of fasting glucose and lipids. The RT-PCR analysis revealed decreased expression of ACE and an increase of ACE2 [Ang-(1–7) marker] in group treated with resveratrol and also an increased expression of SIRT1 in groups that received Ang-(1–7). We showed for the first time that improved metabolic profile is associated with increased expression of GLUT4 and high expression of AMPK/FOXO1/PPAR-γ pathway in adipose-tissue. Finally, adipocyte primary cell-culture incubated with and without sirtuin and Ang-(1–7)/Mas antagonists pointed out for a cross-talking between RAS and sirtuins. We conclude that oral administration of Ang-(1–7) and RSV improved metabolic profile through a cross-modulation between RAS and Sirtuins.
Keywords: Resveratrol; Rennin-angiotensin system; Ang-(1–7); Obesity; Metabolic syndrome;