Peptides (v.56, #C)

Exercise-mimicking treatment fails to increase Fndc5 mRNA & irisin secretion in primary human myotubes by Timea Kurdiova; Miroslav Balaz; Alexander Mayer; Denisa Maderova; Vitazoslav Belan; Christian Wolfrum; Jozef Ukropec; Barbara Ukropcova (1-7).
Irisin, myokine secreted by skeletal muscle, was suggested to mediate some of exercise health benefits via “browning” of white adipose tissue. However, mounting evidence contradicts the regulatory role of exercise for muscle irisin production/secretion in humans. Thus, we explored the direct effect of exercise-mimicking treatment on irisin in human primary muscle cells in vitro. Human primary muscle cell cultures were established from lean, obese prediabetic and type-2-diabetic individuals. Complex metabolic phenotyping included assessment of insulin sensitivity (euglycemic hyperinsulinemic clamp) and adiposity content&distribution (MRI&MRS). In vitro exercise-mimicking treatment (forskolin + ionomycin) was delivered in 1-h pulse/day during differentiation. Fndc5 mRNA (qRT-PCR) and secreted irisin (ELISA) were determined in cells and media. Exercise-mimicking treatment more than doubled Pgc1α mRNA in differentiated muscle cells. Nevertheless, Fndc5 mRNA was reduced by 18% and irisin in media by 20%. Moreover, Fncd5 mRNA was increased in myotubes derived from individuals with type-2-diabetes, independent on exercise-mimicking treatment. Fndc5 mRNA in cells was positively related to fasting glycemia (p  = 0.0001) and negatively to whole-body insulin sensitivity (p  < 0.05). Collectively, our data do not support the role of exercise-related signaling pathways in irisin regulation in human skeletal muscle and confirm our previous observations on increased Fndc5 expression in muscle cells from individuals with type-2-diabetes.
Keywords: Fndc5; Irisin; Myokines; Exercise-mimicking treatment; Human myotubes; Type 2 diabetes;

Tapentadol inhibits calcitonin gene-related peptide release from rat brainstem in vitro by Maria Cristina Greco; Lucia Lisi; Diego Currò; Pierluigi Navarra; Giuseppe Tringali (8-13).
We have previously developed an in vitro model of rat brainstem explants. The latter release sizable amounts of calcitonin gene-related peptide (CGRP); basal release can be stimulated by such secretagogues as high KCl concentrations, veratridine or capsaicine. In this paradigm we investigated the activity of the analgesic agent tapentadol; the effects of tapentadol were compared to those of a classical opioid receptor agonist, morphine, and the selective noradrenaline reuptake inhibitor reboxetine. Morphine inhibited basal CGRP release, with statistical significance from 1 nM onward and maximal (−44%) inhibition at 100 μM. Morphine also inhibited K+-stimulated peptide release, with a significant effect from 1 μM and maximal (−39%) decrease at 100 μM, but failed to inhibit release stimulated by 10 μM capsaicin. At variance, reboxetine had no effect on baseline CGRP outflow, but was able to inhibit both K+-stimulated [significant inhibition from 1 μM onward and maximal (−37%) decrease at 100 μM], and capsaicin-stimulated release [significant effect from 1 μM and maximal (−31%) decrease at 100 μM]. Likewise, tapentadol had no effect on baseline CGRP release up to 100 μM, but decreased secretion stimulated by 56 mM KCl or capsaicin, with significant effects from 0.1 and 1 μM respectively; maximal inhibition over 56 mM KCl and capsaicin stimuli was −29% and −31%, respectively. Naloxone antagonized the effect of morphine, but not those of reboxetine and tapentadol, on K+-stimulated CGRP secretion. In conclusion the present study provides consistent pharmacological evidence that tapentadol acts as a noradrenaline reuptake inhibitor agent in this experimental model.
Keywords: Calcitonin gene-related peptide; Tapentadol; Morphine; Reboxetine; Brainstem; Rat;

(Pro)renin receptor ((P)RR) is a specific receptor for both renin and its precursor prorenin. (P)RR was shown to be involved in pathophysiology of cardiovascular and renal diseases. Soluble (pro)renin receptor (s(P)RR), which is generated by furin from full length (P)RR, is present in blood. The aim of the present study is to clarify the association of plasma s(P)RR levels and the severity of OSAS. Plasma levels of s(P)RR were measured by ELISA in 58 male patients diagnosed as OSAS based on polysomnography, and 14 age-matched male control subjects. Blood samples were obtained at 6:00 a.m. just after overnight polysomnography. Plasma s(P)RR levels were significantly higher in patients with OSAS (9.0 ± 2.0 ng/mL, mean ± SD) than in control subjects (7.4 ± 1.5 ng/mL) (P  = 0.0026). Plasma s(P)RR levels showed a significant negative correlation with % stage rapid eye movement (REM) sleep (r  = −0.377, p  < 0.005), and significant positive correlations with % stage 1 (r  = 0.374, p  < 0.005), arousal index (r  = 0.341, p  < 0.01), apnea hypopnea index (AHI) (r  = 0.352, p  < 0.01) and desaturation index (r  = 0.302, p  < 0. 05). In 12 OSAS patients with AHI ≥20, plasma levels of s(P)RR were studied after 3-month treatment with nasal continuous positive airway pressure (nCPAP). Plasma s(P)RR levels were significantly decreased after the nCPAP treatment (p  = 0.0016). The present study has shown for the first time elevated plasma s(P)RR levels in patients with OSAS. Plasma s(P)RR levels were associated with the severity of OSAS. Soluble (P)RR may serve as a plasma marker reflecting the severity of OSAS.
Keywords: Obstructive sleep apnea syndrome; Soluble (pro)renin receptor; Prorenin; Renin; Aldosterone; Apnea hypopnea index;

Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: Membrane integrity disruption and inhibition of biofilm formation by Kairong Wang; Jiexi Yan; Wen Dang; Junqiu Xie; Bo Yan; Wenjin Yan; Mengyang Sun; Bangzhi Zhang; Mingxia Ma; Yanyan Zhao; Fengjing Jia; Ranran Zhu; Wei Chen; Rui Wang (22-29).
With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC50) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.
Keywords: Antifungal activity; Antimicrobial peptide; Polybia-MPI; Biofilm formation;

Plant peptides in defense and signaling by Nelson Marmiroli; Elena Maestri (30-44).
This review focuses on plant peptides involved in defense against pathogen infection and those involved in the regulation of growth and development. Defense peptides, defensins, cyclotides and anti-microbial peptides are compared and contrasted. Signaling peptides are classified according to their major sites of activity. Finally, a network approach to creating an interactomic peptide map is described.
Keywords: Arabidopsis thaliana; Interactome; Mass spectrometry; Systems biology;

The hypotensive effect of intrathecally injected (m)VD-hemopressin(α) in urethane-anesthetized rats by Xu-hui Li; Ning Li; Zi-long Wang; Jia-xin Pan; Zheng-lan Han; Xue-mei Chang; Hong-hai Tang; Pei Wang; Rui Wang; Quan Fang (45-51).
Previous studies suggest that cannabinoids system plays an important role in cardiovascular regulation. (m)VD-hemopressin(α) (VD-Hpα), an 11-residue peptide originating from the α1 chain of hemoglobin, was recently reported as a selective agonist of cannabinoid CB1 receptor. The present study was undertaken to investigate the intrathecal (i.t.) action of (m)VD-Hpα on blood pressure in urethane-anesthetized rats. Our results demonstrated that injections of (m)VD-Hpα (5–30 nmol, i.t.) produced a dose-dependent decrease in mean arterial pressure (MAP), similar to that of the non-peptidic cannabinoid receptor agonist WIN55212-2 (1.25–10 nmol, i.t.). The hypotensive effect of (m)VD-Hpα was not influenced by the CB1 receptor antagonist AM251 (20 nmol, i.t.) or the CB2 receptor antagonist AM630 (20 nmol, i.t.). However, WIN55212-2-induced hypotension was almost completely prevented by i.t. administration of AM251, not by AM630. The spinal hypotension of (m)VD-Hpα and WIN55212-2 was significantly reduced by pretreatment with the α-adrenoceptor antagonist phentolamine (1 mg/kg, i.v.), but not by the β-adrenoceptor antagonist propranolol (2 mg/kg, i.v.) or the muscarinic receptor antagonist atropine (2 mg/kg, i.v.). In addition, l-NAME (50 mg/kg, i.v.), the inhibitor of nitric oxide (NO) synthase, significantly reduced WIN55212-2-induced hypotension, but had no effect on the hypotensive response to (m)VD-Hpα. Collectively, the results show that i.t. administration of (m)VD-Hpα induces a decrease in MAP via a non-CB1 and non-CB2 mechanism.
Keywords: Cannabinoid; (m)VD-hemopressin(α); Hypotensive response; Antagonist; Rat;

Comparision of secretagogue effects of rosiglitazone and telmisartan on ANP secretion in rats by Shan Gao; Byung Mun Park; Seung Ah Cha; Sung Zoo Kim; Suhn Hee Kim (52-58).
Peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear transcription factor, is a key regulator of insulin signaling, and glucose and fat metabolism. In this study, we evaluated the direct effect of PPAR-γ ligand on the secretion of atrial natriuretic peptide (ANP). The isolated perfused beating atria were used and rosiglitazone (0.01, 0.3 and 1 μM) or telmisartan was perfused into atria with and without inhibitors. High frequency stimulation caused a decreased atrial contractility by 40% and an increased ANP secretion by 80%. Rosiglitazone augmented high frequency-induced ANP secretion and concentration in a dose-dependent manner. Rosiglitazone-induced ANP secretion was attenuated by the pretreatment with PPAR-γ antagonist (GW 9662), or inhibitor for phosphoinositol 3-kinase (PI3-kinase, wortmannin), Akt (API-2) or nitric oxide synthase (l-NAME). Telmisartan, a partial agonist of PPAR-γ with angiotensin II type 1 receptor (AT1R) blocker, also stimulated ANP secretion, which was more potent than rosiglitazone or losartan. Infusion of rosiglitazone or telmisartan in anesthetized rats tended to decrease mean arterial pressure and to increase pulse pressure without difference. A plasma ANP level was increased by telmisartan more than by rosiglitazone. In diabetic rats, an increased plasma ANP level was more prominent than sham rats. Therefore, we suggest that rosiglitazone stimulates high frequency-induced ANP secretion through the PPAR-γ receptor-PI3-kinase-Akt-eNOS pathway and telmisartan shows synergistic effect on ANP secretion.
Keywords: PPAR-γ receptor; Rosiglitazone; Telmisartan; Atrial natriuretic peptide; Receptor;

Age versus nutritional state in the development of central leptin resistance by Erika Pétervári; Ildikó Rostás; Szilvia Soós; Judit Tenk; Alexandra Mikó; Nóra Füredi; Miklós Székely; Márta Balaskó (59-67).
Leptin, a catabolic adiposity signal acts in the hypothalamus via suppressing food intake and inducing hypermetabolism. Age and obesity are accompanied by leptin resistance. The present study aimed to clarify which components of the catabolic leptin effects are influenced most strongly by aging and which ones by nutritional state-induced alterations in body composition. In our biotelemetric study the effects of a 7-day intracerebroventricular leptin infusion on various parameters of energy balance (food intake, body weight, oxygen consumption, heart rate and body temperature) were analyzed in male Wistar rats of different age-groups (from 3 to 24 months) and nutritional states (normally fed, diet-induced obese and calorie-restricted). Leptin resistance of older animals affected hypermetabolic actions, whereas leptin induced anorexia in all age-groups. Weight reducing effect of leptin diminished in middle-aged and aging animals to become significant again in the oldest group. In diet-induced obese rats leptin-induced hypermetabolism of the young rats and hypermetabolism plus anorexia of the aging ones were suppressed. Calorie-restriction reduced body weight and fat mass to a similar extent in all age-groups. It strongly enhanced leptin-induced hypermetabolism at all ages and prevented the manifestation of anorexigenic actions of leptin with the exception of the oldest group. This latter finding suggests an unexpected increase of responsiveness to anorexigenic leptin actions in old rats. Accordingly, anorexia and hypermetabolism change in disparate ways with aging. Nutritional state predominantly influences hypermetabolic leptin actions. Resistance to both hypermetabolic and anorexigenic actions were promoted by obesity, while calorie-restriction enhanced responsiveness to leptin, especially in old rats.
Keywords: Leptin; Aging; Obesity; Calorie-restriction; Energy balance;

Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity by Oleg Sokolov; Natalya Kost; Olga Andreeva; Ekaterina Korneeva; Viktor Meshavkin; Yulia Tarakanova; Aleksander Dadayan; Yurii Zolotarev; Sergei Grachev; Inna Mikheeva; Oleg Varlamov; Andrey Zozulya (68-71).
Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.
Keywords: Autism; Urine; Bovine casomorphin; Opioid peptides;

The relation of leptin and soluble leptin receptor levels with metabolic and clinical parameters in obese and healthy children by Gonul Catli; Ahmet Anik; Hale Ünver Tuhan; Tuncay Kume; Ece Bober; Ayhan Abaci (72-76).
We investigated the relation of serum leptin, soluble leptin receptor (sLR) and free leptin index (FLI) with metabolic and anthropometric parameters in obese and healthy children. Height, weight, waist circumference (WC), fasting serum glucose, insulin, lipid profile, leptin and sLR levels of 35 obese children and 36 healthy children were measured and FLI was calculated as the ratio of leptin to sLR. In obese children, serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children. Comparison of obese children regarding the insulin resistance showed significantly higher serum leptin and FLI in the insulin resistant group; however sLR level was not different between the insulin resistant and non-resistant obese children. In obese children, sLR was not correlated with any of the metabolic parameters except total cholesterol, while FLI was significantly and positively correlated with BMI, WC, TC, fasting insulin, and HOMA-IR. However, regression analysis confirmed that the HOMA-IR was the only independent variable significantly correlated with FLI in obese children. Findings of this study suggest that in obese children and adolescents (i) serum leptin and FLI were found significantly higher, while sLR level was significantly lower than the healthy children, (ii) increased FLI might be a compensatory mechanism for increasing leptin effect in peripheral tissue, (iii) FLI is a more accurate marker to evaluate leptin resistance than leptin or sLR alone, and (iv) increased FLI may contribute toward the development of hyperinsulinemia and insulin resistance.
Keywords: Childhood; Obesity; Leptin; Soluble leptin receptor; Insulin resistance;

Soluble expression, purification and functional identification of the framework XV conotoxins derived from different Conus species by Yun Wu; Lei Wang; Maojun Zhou; Xiuhua Jiang; Xiaoyan Zhu; Yu Chen; Shaonan Luo; Yuwen You; Zhenghua Ren; Anlong Xu (77-83).
The conotoxin cysteine framework XV (-C-C-CC-C-C-C-C-), which was named Lt15a, was firstly identified from the cDNA library of Conus litteratus. After that, 18 new framework XV conotoxin sequences were cloned from nine Conus species. Like other conopeptides, the XV-conotoxins have the conserved signal peptide and propeptide, and there are also some conserved residues in their mature peptide. All the framework XV conotoxins were apparently converged into two branches, because of the indel and point mutations occurred in their mature peptides. By fused with thioredoxin and 6 × His tag, six XV-conotoxins were successfully expressed in Escherichia coli and purified. Different framework XV conotoxins have distinct biological activities on mice and frogs, and that may be related to the diversity of the toxin sequences. All the six XV-conotoxins had no obvious effects on the sodium currents of DRG neuron cells of Sprague–Dawley (SD) rats. The identification of this framework of conotoxins enriches our understanding of the structural and functional diversity of conotoxin.
Keywords: Bio-activity; Conotoxin; Cysteine framework; Diversity; Recombinant expression;

Identification of multifunctional peptides from human milk by Santi M. Mandal; Rashmi Bharti; William F. Porto; Samiran S. Gauri; Mahitosh Mandal; Octavio L. Franco; Ananta K. Ghosh (84-93).
Pharmaceutical industries have renewed interest in screening multifunctional bioactive peptides as a marketable product in health care applications. In this context, several animal and plant peptides with potential bioactivity have been reported. Milk proteins and peptides have received much attention as a source of health-enhancing components to be incorporated into nutraceuticals and functional foods. By using this source, 24 peptides have been fractionated and purified from human milk using RP-HPLC. Multifunctional roles including antimicrobial, antioxidant and growth stimulating activity have been evaluated in all 24 fractions. Nevertheless, only four fractions show multiple combined activities among them. Using a proteomic approach, two of these four peptides have been identified as lactoferrin derived peptide and kappa casein short chain peptide. Lactoferrin derived peptide (f8) is arginine-rich and kappa casein derived (f12) peptide is proline-rich. Both peptides (f8 and f12) showed antimicrobial activities against both Gram-positive and Gram-negative bacteria. Fraction 8 (f8) exhibits growth stimulating activity in 3T3 cell line and f12 shows higher free radical scavenging activity in comparison to other fractions. Finally, both peptides were in silico evaluated and some insights into their mechanism of action were provided. Thus, results indicate that these identified peptides have multiple biological activities which are valuable for the quick development of the neonate and may be considered as potential biotechnological products for nutraceutical industry.
Keywords: Antimicrobial peptides; Antioxidant peptides; Growth stimulating peptides; Human milk; Multifunctional peptides;

Homeostasis of energy is regulated by genetic factors, food intake, and energy expenditure. When energy input is greater than expenditure, the balance is positive, which can lead to weight gain and obesity. When the balance is negative, weight is lost. Regulation of this homeostasis is multi-factorial, involving many orexigenic (appetite-stimulating) and anorexigenic (appetite-suppressing) peptide hormones. Peripheral tissues are now known to be involved in weight regulation and research on its endocrine characteristics proceeds apace. Preptin with 34 amino acids (MW 3948 Da), adropin with 43 amino acids and a molecular weight of (4999 Da), and irisin with 112 amino acids (12587 Da), are three newly discovered peptides critical for regulating energy metabolism. Preptin is synthesized primarily in pancreatic beta cells, and adropin mainly in the liver and brain, and many peripheral tissues. Irisin, however, is synthesized principally in the heart muscle, along with peripheral tissues, including salivary glands, kidney and liver. The prime functions of preptin and adropin include regulating carbohydrate, lipid and protein metabolisms by moderating glucose-mediated insulin release. Irisin is an anti-obesitic and anti-diabetic hormone regulating adipose tissue metabolism and glucose homeostasis by converting white to brown adipose tissue. This review offers a historical account of these discovery and function of these peptides, including their structure, and physiological and biochemical properties. Their roles in energy regulation will be discussed. Their measurement in biological fluids will be considered, which will lead to further discussion of their possible clinical value.
Keywords: Preptin; Adropin; Irisin; Energy regulation;

High plasma proenkephalin A level has been associated with ischemic stroke severity and clinical outcomes. This study aimed to assess the relationship between proenkephalin A and disease severity as well as to investigate its ability to predict long-term clinical outcome in patients with aneurysmal subarachnoid hemorrhage. Plasma proenkephalin A concentrations of one hundred and eighty patients and 180 sex- and age-matched healthy controls were measured by chemoluminescence sandwich immunoassay. Plasma proenkephalin A level was substantially higher in patients than in healthy controls (205.5 ± 41.6 pmol/L vs. 90.8 ± 21.1 pmol/L, P  < 0.001), was highly associated with World Federation of Neurological Surgeons (WFNS) score (r  = 0.470, P  < 0.001) and Fisher score (r  = 0.488, P  < 0.001), was an independent predictor for 6-month mortality [odds ratio (OR), 1.183; 95% confidence interval (CI), 1.067–1.339; P  = 0.004] and unfavorable outcome (Glasgow Outcome Scale score of 1–3) (OR, 1.119; 95% CI, 1.046–1.332; P  = 0.005) using multivariate analysis, and had high area under receiver operating characteristic curve (AUC) for prediction of 6-month mortality (AUC, 0.831; 95% CI, 0.768–0.883) and unfavorable outcome (AUC, 0.821; 95% CI, 0.757–0.874). The predictive value of the plasma proenkephalin A concentration was also similar to those of WFNS score and Fisher score (both P  > 0.05). In a combined logistic-regression model, proenkephalin A improved the AUCs of WFNS score and Fisher score, but the differences were not significant (both P  > 0.05). Thus, proenkephalin A level may be a useful, complementary tool to predict mortality and functional outcome at 6 months after aneurysmal subarachnoid hemorrhage.
Keywords: Proenkephalin A; Aneurysmal subarachnoid hemorrhage; Mortality; Functional outcome;

cDNAs encoding the appetite regulating peptides apelin, cocaine and amphetamine regulated transcript (CART), cholecystokinin (CCK), peptide YY (PYY) and orexin were isolated in red-bellied piranha and their mRNA tissue and brain distributions examined. When compared to other fish, the sequences obtained for all peptides were most similar to that of other Characiforme fish, as well as to Cypriniformes. All peptides were widely expressed within the brain and in several peripheral tissues, including gastrointestinal tract. In order to assess the role of these peptides in the regulation of feeding of red-bellied piranha, we compared the brain mRNA expression levels of these peptides, as well as the gut mRNA expression of CCK and PYY, between fed and 7-day fasted fish. Within the brain, fasting induced a significant increase in both apelin and orexin mRNA expressions and a decrease in CART mRNA expression, but there where were no significant differences for either PYY or CCK brain mRNA expressions between fed and fasted fish. Within the intestine, PYY mRNA expression was lower in fasted fish compared to fed fish but there was no significant difference for CCK intestine mRNA expression between fed and fasted fish. Our results suggest that these peptides, perhaps with the exception of CCK, play a major role in the regulation of feeding of red-bellied piranha.
Keywords: Red-bellied piranha; Apelin; CART; CCK; PYY; Orexin; Feeding; Fasting; mRNA expression;

Hemopressin, an inverse agonist of cannabinoid receptors, inhibits neuropathic pain in rats by Elaine F. Toniolo; Estêfani T. Maique; Wilson A. Ferreira; Andrea S. Heimann; Emer S. Ferro; Dinah L. Ramos-Ortolaza; Lydia Miller; Lakshmi A. Devi; Camila S. Dale (125-131).
Direct-acting cannabinoid receptor ligands are well known to reduce hyperalgesic responses after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Hemopressin (Hp) is a nonapeptide that selectively binds CB1 cannabinoid receptors (CB1 receptors) and exerts antinociceptive action in inflammatory pain models. We investigated the effect of Hp on neuropathic pain in rats subjected to chronic constriction injury (CCI) of the sciatic nerve, and explored the mechanisms involved. Oral administration of Hp inhibits mechanical hyperalgesia of CCI-rats up to 6 h. Hp treatment also decreases Egr-1 immunoreactivity (Egr-1Ir) in the superficial layer of the dorsal horn of the spinal cord of CCI rats. The antinociceptive effect of Hp seems to be independent of inhibitory descending pain pathway since methysergide (5HT1A receptor antagonist) and yohimbine (α-2 adrenergic receptor antagonist) were unable to prevent Hp antinociceptive effect. Hp decreased calcium flux on DRG neurons from CCI rats, similarly to that observed for AM251, a CB1 receptor antagonist. We also investigated the effect of Hp on potassium channels of CCI rats using UCL 1684 (a blocker of Ca2+-activated K+ channels) which reversed Hp-induced antinociception. Furthermore, concomitant administration of URB-584 (FAAH inhibitor) but not JZL-184 (MAGL inhibitor) potentiates antinociceptive effect of Hp in CCI rats indicating an involvement of anadamide on HP-induced antinociception. Together, these data demonstrate that Hp displays antinociception in pain from neuropathic etiology through local effects. The release of anandamide and the opening of peripheral K+ channels are involved in the antinociceptive effect.
Keywords: CB1 cannabinoid receptors; Hemopressin; Chronic constriction injury model; Hyperalgesia; Dorsal root ganglion;

A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae) by J. Michael Conlon; Milena Mechkarska; Gordana Radosavljevic; Samir Attoub; Jay D. King; Miodrag L. Lukic; Stephen McClean (132-140).
Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH2) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH2) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16 μM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC50  = 80 ± 6 μM) and 2.2S (LC50  = 75 ± 5 μM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC50  = 167 ± 8 μM for frenatin 2.1S and 169 ± 7 μM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P  < 0.05) increased production of proinflammatory cytokines IL-1β and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.
Keywords: Frog skin; Frenatin; Hylidae; Antimicrobial; Immunomodulatory; Cytokine;

Decreased saliva/serum irisin concentrations in the acute myocardial infarction promising for being a new candidate biomarker for diagnosis of this pathology by Suna Aydin; Suleyman Aydin; Mehmet Ali Kobat; Mehmet Kalayci; Mehmet Nesimi Eren; Musa Yilmaz; Tuncay Kuloglu; Evrim Gul; Ozlem Secen; Omer Dogan Alatas; Adil Baydas (141-145).
Irisin is a muscle-secreted protein. Cardiac muscle produces more irisin than skeletal muscle in response to acute exercise, and is associated with myocardial infarction (MI) in an experimental model induced by isoproterenol in rats. The timing and significance of its release in patients with acute myocardial infarction (AMI) needs further investigation. We have studied the relationship between serum/saliva irisin concentration and AMI in humans. Serum and saliva samples were taken within 3 days of admission in 11 patients with AMI and in 14 matched controls. Salivary gland irisin was detected immunohistochemically, and serum and saliva levels were measured by ELISA. The three major paired salivary glands (submandibular, sublingual and parotid) produce and release irisin into saliva. Troponin-I, CK, CK-MB concentrations in the AMI group gradually increased from up to 12 h, while saliva and serum irisin gradually decreased from up to 48 h, compared with the control group (P  < 0.05). After 12 h, troponin-I, CK, CK-MB started to decrease, while saliva and serum irisin started to increase at 72 h. Serum irisin levels correlated with age, while troponin I, CK-MB, and CK were correlated and with saliva irisin in AMI patients. Besides cardiac troponin and CK-MB, irisin adds new diagnostic information in AMI patients, and the gradual decrease of saliva/serum irisin over 48 h could be a useful biomarker.
Keywords: Irisin; Myocardial infarction; Saliva; Serum;

High plasma levels of adrenomedullin have been associated with stroke severity and clinical outcomes. This study aimed to analyze plasma levels of adrenomedullin in traumatic brain injury and their association with prognosis. One hundred and forty-eight acute severe traumatic brain injury and 148 sex- and age-matched healthy controls were recruited in this study. Plasma adrenomedullin concentration was measured by enzyme-linked immunosorbent assay. Unfavorable outcome was defined as Glasgow Outcome Scale score of 1–3. Compared to controls, the patients had significantly higher plasma concentrations of adrenomedullin, which were also highly associated negatively with Glasgow Coma Scale score. Plasma adrenomedullin level was proved to be an independent predictor for 6-month mortality and unfavorable outcome of patients in a multivariate analysis. A receiver operating characteristic curve was configured to show that a baseline plasma adrenomedullin level predicted 6-month mortality and unfavorable outcome of patients with high area under curve. The predictive performance of the plasma adrenomedullin concentration was also similar to that of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients. In a combined logistic-regression model, adrenomedullin improved the area under curve of Glasgow Coma Scale score for the prediction of 6-month mortality and unfavorable outcome of patients, but the differences did not appear to be statistically significant. Thus, high plasma levels of adrenomedullin are associated with head trauma severity, and may independently predict long-term clinical outcomes of traumatic brain injury.
Keywords: Adrenomedullin; Traumatic brain injury; Mortality; Functional outcome;

Urinary tract infections (UTI) are important health problems and predisposing causes of UTI are not entirely known. Neuro-immune interactions play an important role in human health and disease. Capsaicin-sensitive sensory nerves which in nerve bladder extensively regulate immune system through neuropeptides such as substance P (SP), calcitonin-gene related peptide (CGRP) and vasoactive intestinal peptide (VIP). In addition these neuropeptides also have anti-bacterial effects. To determine how the levels of these peptides changes during UTI, 67 patients (50–90 years-old) diagnosed with UTI in Akdeniz University Faculty of Medicine Hospital were compared with 37 healthy people 50 years or older as the control group. Additionally, 7 patients with UTI symptoms (dysuria, urgency) but with sterile pyuria were also included in the study. Urine samples from 15 patients, whose symptoms regressed with control urine cultures being sterile, were taken after completion of the treatments. Urine neuropeptide levels were determined by ELISA. CGRP levels are significantly higher in patients with UTI, but did not associate with pyuria whereas SP and VIP levels were significantly lower in patients with sterile pyuria, indicating sensory nerve deficiency. Since CGRP exerts immunosuppressive effects, increased levels of the peptide may predispose to UTI. Furthermore, the connection between the observed sensory nerve deficiency and sterile pyuria warrants further studies.
Keywords: Urinary tract infections; Substance P; Vasoactive intestinal peptide; Calcitonin gene related peptide; Sterile pyuria; Capsaicin;

One dose of oral hexarelin protects chronic cardiac function after myocardial infarction by Yuanjie Mao; Takeshi Tokudome; Ichiro Kishimoto; Kentaro Otani; Mikiya Miyazato; Kenji Kangawa (156-162).
Both hexarelin and its natural analog ghrelin exert comparable cardioprotective activities. A single dose of ghrelin administered at the very acute phase after experimental myocardial infarction positively affects cardiac function in chronic heart failure. Therefore, this study aimed to determine whether a single dose of oral hexarelin has the same effect in the chronic disease phase. Myocardial infarction or sham operation was generated by left coronary artery ligation in male C57BL/6J mice, which subsequently received one dose of hexarelin or vehicle treatment by oral gavage 30 min after operation. Although the mortality within 14 days after myocardial infarction did not differ between the groups, hexarelin treatment protected cardiac function in the chronic phase as evidenced by higher ejection fraction and fractional shortening, as well as lower lung weight/body weight and lung weight/tibial length ratios, compared with vehicle treatment. Hexarelin treatment concurrently lowered plasma epinephrine and dopamine levels, and shifted the balance of autonomic nervous activity toward parasympathetic nervous activity as evidenced by a smaller low/high-frequency power ratio and larger normalized high-frequency power on heart rate variability analysis. The results first demonstrate that one dose of oral hexarelin treatment potentially protects chronic cardiac function after acute myocardial infarction, and implicate that activating growth hormone secretagogue receptor 1a might be beneficial for cardioprotection, although other mechanism may also be involved.
Keywords: Hexarelin; Cardiac function; Myocardial infarction; Sympathetic nerve;

Alarin is a newly identified member of the galanin family of peptides. Galanin has been shown to exert regulatory effects on depression. Similar to galanin in distribution, alarin is also expressed in the medial amygdala and hypothalamus, i.e., regions interrelated with depression. However, it remains a puzzle whether alarin is involved in depression. Accordingly, we established the depression-like mouse model using behavioral tests to ascertain the possible involvement of alarin, with fluoxetine as a positive control. With the positive antidepressant-like effects of alarin, we further examined its relationship to HPA axis activity and brain-derived neurotrophic factor (BDNF) levels in different brain areas in a chronic unpredictable mild stress (CUMS) paradigm. In the acute studies, alarin produced a dose-related reduction in the immobility duration in tail suspension test (TST) in mice. In the open-field test, intracerebroventricular (i.c.v.) injection of alarin (1.0 nmol) did not impair locomotion or motor coordination in the treated mice. In the CUMS paradigm, alarin administration (1.0 nmol, i.c.v.) significantly improved murine behaviors (FST and locomotor activity), which was associated with a decrease in corticotropin-releasing hormone (CRH) mRNA levels in the hypothalamus, as well as a decline in serum levels of CRH, adrenocorticotropic hormone (ACTH) and corticosterone (CORT), all of which are key hormones of the HPA axis. Furthermore, alarin upregulated BDNF mRNA levels in the prefrontal cortex and hippocampus. These findings suggest that alarin may potentiate the development of new antidepressants, which would be further secured with the identification of its receptor(s).
Keywords: Alarin; Depression; Hypothalamic–pituitary–adrenal axis; Brain-derived neurotrophic factor;

Effects of urotensin II on intracellular pH regulation in cultured human internal mammary artery smooth muscle cells by Yi-Ting Tsai; Chung-Yi Lee; Chih-Chin Hsu; Chung-Yi Chang; Ming-Kai Hsueh; Eagle Yi-Kung Huang; Chien-Sung Tsai; Shih-Hurng Loh (173-182).
The Na+–H+ exchanger (NHE) and the Na+–HCO3 co-transporter (NBC) have been confirmed as two major active acid extruders in many mammalian cells. Whether the NHE and NBC functional co-exist in human internal mammary artery smooth muscle cells (HIMASMCs) remains unclear. The aims of the present study were to investigate the acid-extruding mechanisms and to explore the effects of urotensin-II (U-II), a powerful vasoconstrictor, on pHi regulators in HIMASMCs. We investigated the changes of pHi by BCECF-fluorescence in HIMASMCs. We found that (a) two Na+-dependent acid extruders, i.e. NHE and NBC, functionally co-exist; (b) U-II (3–100 nM) induced a concentration-dependent intracellular acidosis; and (c) U-II (3–100 nM) caused a concentration-dependent increase on NHE activity, while decrease on NBC activity. In summary, we demonstrate for the first time that two acid-extruders, NHE and NBC, functionally co-exist in HIMASMCs. Moreover, U-II induces a concentration-dependent intracellular acidosis through the balanced effect of its effect on increasing NHE activity and decreasing NBC activity.
Keywords: Intracellular pH; Human internal mammary artery smooth muscle cells; Fluorescent-BCECF; Urotensin-II (U-II); Na+–H+ exchanger (NHE); Na+–HCO3 co-transporter (NBC);