Peptides (v.28, #5)

Regulation of hepcidin in HepG2 and RINm5F cells by Evelyn Fein; Uta Merle; Robert Ehehalt; Thomas Herrmann; Hasan Kulaksiz (951-957).
Despite the high impact of the antimicrobial peptide hepcidin in iron homeostasis, the regulation of this hormone is still not completely understood. Studies concerning hepcidin regulation are performed at the mRNA level. For the first time we analyzed the regulation of hepcidin not only at mRNA, but also at protein level in a hepatoma and a pancreatic beta cell line using quantitative RT-PCR and immunoblot analysis. Our data show, that hepcidin is present in HepG2 and RINm5F cells. A significant up-regulation of hepcidin was observed in both cell lines by the inflammatory cytokine interleukin-6, lipopolysaccharide, and a slight upregulation by deferoxamine. A down-regulation was detected after stimulation with erythropoietin. Hepcidin was regulated by iron in a dose dependent manner: low doses up to 3 μM increased hepcidin expression, high doses of iron (65 μM) revealed a switch-over to down-regulation of hepcidin expression. Regulation of hepcidin in HepG2 and RINm5F cells at mRNA and protein level by these substances indicates its involvement in inflammation and iron metabolism.
Keywords: Hepcidin; Pro-hepcidin; Iron metabolism; Pancreas; Liver;

Conjugation of a novel Apaf-1 inhibitor to peptide-based cell-membrane transporters: by Mar Orzáez; Laura Mondragón; Isabel Marzo; Glòria Sanclimens; Àngel Messeguer; Enrique Pérez-Payá; María J. Vicent (958-968).
We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.
Keywords: Apoptosis; Apoptosome inhibitors; Cell-penetrating peptides; Drug delivery systems; Conformational analysis; Membrane mimetics;

A bactericidal homodimeric phospholipases A2 from Bungarus fasciatus venom by Chunhua Xu; Dongying Ma; Haining Yu; Zongjie Li; Jianguo Liang; Guoqing Lin; Yun Zhang; Ren Lai (969-973).
Group IIA secretory phospholipases A2 (sPLA2-II) is generally known to display potent gram-positive bactericidal activity, while group IA sPLA2 (sPLA2-I) reportedly is not. In this work, a novel sPLA2-I named BFPA was identified from Bungarus fasciatus venom, and its antimicrobial activity was studied as well. The amino acid sequence of the venomous protein precursor was 145-amino acid in length, and contained a predicted 27-amino acid signal peptide and a 118-amino acid mature protein. Unlike the well-known sPLA2-Is, which have 14 half-cysteines forming 7 intramolecular disulfide bridges, BFPA possesses 15 half-cysteines. The additional cysteine might contribute to the formation of an intermolecular disulfide bridge of the homodimeric protein. In the biological activities assays, BFPA displayed the activities of anticoagulation and bactericidal against Escherichia coli and Staphylococcus aureus. This study is the first report about gram-positive bactericidal activity of sPLA2-I.
Keywords: PLA2; Bungarus fasciatus; Antibacterial; Snake venom;

The level of adipokinetic hormones (AKHs) (Peram-CAH-I and II) in the corpora cardiaca and the hemolymph of Leptinotarsa decemlineata enormously increases in the adults fed on genetically modified potatoes containing either GNA lectin or Cry 3Aa toxin concomitant with increased oxidative stress in gut tissues. A similar enhancement of the AKH titer is achieved when the adults are injected with paraquat that evokes oxidative stress. On the other hand, an injection of exogenous AKH reduces oxidative stress biomarkers in the hemolymph by reducing protein carbonyls and enhancing reduced glutathione levels. These facts indicate that there is a feedback regulation between an oxidative stressor action and the level of AKH in the insect body, and that AKHs might be involved in the activation of an antioxidant protection mechanism. These results are to our knowledge, the first evidence for the involvement of AKHs in oxidative stress mitigation, in addition to a plethora of other roles.
Keywords: Adipokinetic hormone; Oxidative stress; GMO; GNA; Cry 3Aa; Paraquat;

Obestatin is a recently discovered peptide hormone that appears to be involved in reducing food intake, gut motility and body weight. Obestatin is a product of the preproghrelin gene and appears to oppose several physiological actions of ghrelin. This study investigated the acute effects of obestatin (1–23) and the truncated form, obestatin (11–23), on feeding activity, glucose homeostasis or insulin secretion. Mice received either intraperitoneal obestatin (1–23) or (11–23) (1 μmol/kg) 4 h prior to an allowed 15 min period of feeding. Glucose excursions and insulin responses were lowered by 64–77% and 39–41%, respectively, compared with saline controls. However this was accompanied by 43% and 53% reductions in food intake, respectively. The effects of obestatin peptides were examined under either basal or glucose (18 mmol/kg) challenge conditions to establish whether effects were independent of changes in feeding. No alterations in plasma glucose or insulin responses were observed. In addition, obestatin peptides had no effect on insulin sensitivity as revealed by hypoglycaemic response when co-administered with insulin. Our observations support a role for obestatin in regulating metabolism through changes of appetite, but indicate no direct actions on glucose homeostasis or insulin secretion.
Keywords: Obestatin; Food intake; Glucose homeostasis; Insulin secretion; Insulin action;

Interoceptive “satiety” signals produced by leptin and CCK by Scott E. Kanoski; Elwood K. Walls; T.L. Davidson (988-1002).
The present studies assessed the extent to which the adiposity signal leptin and the brain–gut hormone cholecystokinin (CCK), administered alone or in combination, give rise to interoceptive sensory cues like those that are produced by a low (1 h) level of food deprivation. Rats were trained with cues arising from 1 to 24-h food deprivation as discriminative stimuli. For one group, 24-h food deprivation predicted the delivery of sucrose pellets, whereas 1-h food deprivation did not. Another group received the reversed deprivation level-sucrose contingency. After asymptotic performance was achieved, the effects of leptin and CCK on food intake and on discrimination performance were tested under 24-h food deprivation. In Experiment 1a, leptin administered into the third cerebroventricle (i3vt) at 3.5 or 7.0 μg doses had little effect, compared to saline on food intake or discriminative responding. In Experiment 1b, leptin (7.0 μg, i3vt) combined with CCK-8 (2 μg/kg, i.p.) reduced food intake significantly, but the findings indicated that CCK-8 alone produces interoceptive discriminative cues more like those produced by 1- than 24-h food deprivation. Experiment 2a tested rats with i.p. leptin (0.3 and 0.5 mg/kg). Although neither dose suppressed intake, the 0.3 mg/kg dose produced interoceptive cues like 1-h food deprivation. Experiment 2b tested two doses of CCK-8 (2 and 4 mg/kg, i.p.) and found significant intake suppression and generalization of discrimination with both doses of CCK-8. These findings suggest a role for both leptin and CCK in the production of sensory consequences that correspond to “satiety”.
Keywords: Adiposity signal; Discrimination; Energy regulation; Rat;

PEGylation of cholecystokinin prolongs its anorectic effect in rats by Fabián León-Tamariz; Isabelle Verbaeys; Maurits Van Boven; Marcel De Cuyper; Johan Buyse; Elke Clynen; Marnix Cokelaere (1003-1011).
The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6 h reaction at 20 °C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1–2 h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6 h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8 h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.
Keywords: Cholecystokinin; PEGylation; Food intake; Appetite suppression;

Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice by Urszula T. Iwaniec; Stéphane Boghossian; Paul D. Lapke; Russell T. Turner; Satya P. Kalra (1012-1019).
Skeletal growth is tightly coupled to energy balance via complex and incompletely understood mechanisms. Leptin-deficient ob/ob mice are obese and develop multiple pathologies associated with the metabolic syndrome. Additionally, ob/ob mice have skeletal abnormalities. The objective of this study was to evaluate the effects of leptin deficiency and long duration selective central leptin repletion via recombinant adeno-associated virus-leptin (rAAV-lep) gene therapy on bone in growing ob/ob mice. The ob/ob mice were injected in the hypothalamus with either rAAV-lep or rAAV-GFP (control vector). Treated ob/ob and untreated wild-type (WT) mice were then maintained on a normal diet for 15 weeks. In a second experiment, similarly treated mice along with a group of pair-fed mice were maintained for 30 weeks. Leptin was not detected in blood of either rAAV-lep- or rAAV-GFP-treated mice although rAAV-lep-treated mice displayed leptin transgene expression in the hypothalamus. As expected, rAAV-lep normalized body weight and food intake. Compared to WT mice, rAAV-GFP-treated ob/ob mice had decreased femoral length (by 1.6 mm or 10%, P  < 0.001), decreased total femur bone volume (by 3.3 mm3 or 19%, P  < 0.001), but increased cancellous bone volume in the distal femur (by 0.04 mm3 or 60%, P  < 0.09) and lumbar vertebrae (by 0.26 mm3 or 118%, P  < 0.001). Treatment with rAAV-lep rescued the ob/ob skeletal phenotype by increasing femoral length and total bone volume, and decreasing femoral and vertebral cancellous bone volume, so that at 15 weeks post-rAAV-lep injection the ob/ob mice no longer differed from WT mice. No further skeletal changes in either the femur or lumbar vertebra were observed at 30 weeks post-rAAV-lep administration. The results suggest that hypothalamic leptin functions as an essential permissive factor for normal bone growth.
Keywords: Osteoporosis; Peak bone mass; μCT; Obesity;

Potent and selective agonists of α-melanotropin (αMSH) action at human melanocortin receptor 5; linear analogs of α-melanotropin by Maria A. Bednarek; Tanya MacNeil; Rui Tang; Tung M. Fong; M. Angeles Cabello; Marta Maroto; Ana Teran (1020-1028).
α-Melanotropin, Ac-Ser1-Tyr-Ser-Met-Glu-His6-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13-NH2 1, is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of αMSH, including a broadly used and more potent the NDP-αMSH peptide, Ac-Ser1-Tyr-Ser-Nle4-Glu-His6-D-Phe7-Arg8-Trp9-Gly-Lys-Pro-Val13-NH2, are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-αMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand αMSH, are exemplified by compound 7, Ac-Ser1-Tyr-Ser-Met-Glu-Oic6-D-4,4′-Bip7-Pip8-Trp9-Gly-Lys-Pro-Val13-NH2 (Oic: octahydroindole-2-COOH, 4,4′-Bip: 4,4′-biphenylalanine, Pip: pipecolic acid), shortly NODBP-αMSH, which has an IC50  = 0.74 nM (binding assay) and EC50  = 0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-αMSH: Ac-Nle-Glu-Oic6-D-4,4′-Bip7-Pip8-Trp9 -NH2 (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic6-D-4,4′-Bip7-Pip8 segment was found to be critical for high agonist potency, while the C-terminal Trp9 residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.
Keywords: α-Melanotropin; αMSH; Melanocortin peptide; NDP-αMSH; Melanocortin receptor 5; Agonist; Binding affinity; cAMP accumulation assay;

Intra CA1 insulin microinjection improves memory consolidation and retrieval by M. Moosavi; N. Naghdi; S. Choopani (1029-1034).
Although the brain was considered as an insulin-insensitive organ, recent studies have shown that insulin receptors exist in the brain and insulin modulates some of the brain tasks. Insulin and its receptor are found in specific areas of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. The improving effect of insulin on spatial memory acquisition has been shown. In the present study, the effect of insulin microinjection into the CA1 region of rat hippocampus on spatial memory consolidation and retrieval has been investigated. Insulin in 12 MU (but not in 0.5 and 6 MU) improved both memory retrieval and consolidation.
Keywords: Insulin; CA1; Retrieval; Consolidation; Morris water maze; Memory; Rat;

The ability of acute exercise to stimulate appetite and food intake depends on intensity, duration, and agouti-related protein (AGRP) levels. Fasting, as well as any negative energy balance, has been reported to increase AGRP expression in the arcuate nucleus (ARC) of the hypothalamus and other extra-hypothalamic tissues in human and rats. The purpose of the present study was to investigate the response of plasma AGRP, GH and insulin to a single circuit-resistance exercise. Twenty volunteer male college students completed a single bout of circuit-resistance training (10 exercises at 35% of 1RM). Blood samples were collected before, immediately and 30 min following the exercise protocol. Plasma AGRP and GH levels showed a significant increase immediately after exercise and returned to pre exercise values during the recovery period. The data indicate that exercise protocol was able to increase plasma AGRP and GH levels. A higher plasma AGRP level might result in an acute exercise-induced hyperphagia and help to fuel post-exercise restoration processes.
Keywords: AGRP; GH; Insulin; Circuit-resistance exercise;

Lack of potentiation of bradykinin by angiotensin-(1-7) in a type 2 diabetes model: Role of insulin by Viviani Milan Ferreira Rastelli; Maria Aparecida Oliveira; Rosangela dos Santos; Rita de Cássia Tostes Passaglia; Dorothy Nigro; Maria Helena Catelli de Carvalho; Zuleica Bruno Fortes (1040-1049).
Considering the growing importance of the interaction between components of kallikrein-kinin and renin–angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems in an insulin resistance model of diabetes, and the effect of insulin on it. For this the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. Though capable of potentiating BK in non-diabetic rats, Ang-(1-7) did not potentiate BK in n-STZ rats. Chronic but not acute insulin treatment restored the potentiation. This restorative effect of insulin was abolished by a K+ channel blocker (tetraethylammonium), by nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester) and by a cyclooxygenase inhibitor (indomethacin). On the other hand, Na+-,K+-ATPase inhibition (by ouabain) did not abolish the effect of insulin. There was no difference in mRNA and protein expression of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Insulin treatment did not alter the kinin receptor expression. Our data allow us to conclude that diabetes impaired the interaction between BK and Ang-(1-7) and that insulin restores it. The restoring effect of insulin depends on membrane hyperpolarization, nitric oxide release and cyclooxygenease metabolites but not Na+K+-ATPase. Alteration of kinin receptor expression might not be involved in the restoring effect of insulin on the potentiation of BK by Ang-(1-7).
Keywords: Type 2 diabets; Bradykinn; Angiotensin-(1-7)-insulin;

TNF-α acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient—Effects on leptin and insulin signaling pathways by Talita Romanatto; Maristela Cesquini; Maria E. Amaral; Érika A. Roman; Juliana C. Moraes; Márcio A. Torsoni; Ariovaldo P. Cruz-Neto; Lício A. Velloso (1050-1058).
Acting in the hypothalamus, tumor necrosis factor-α (TNF-α) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-α to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-α promotes a reduction of 25% in 12 h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-α increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-α, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NFκB, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-α activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-α, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-α is employed. In conclusion, TNF-α, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus.
Keywords: Feeding; Insulin; Leptin; Cytokine; Inflammation;

Inhibitory effects of glucocorticoids on urocortin-mediated increases in interleukin-6 gene expression in rat aortic smooth muscle cells by Kazunori Kageyama; Komaki Hanada; Takeshi Nigawara; Ken-Ichi Furukawa; Ken Terui; Eriko Ogura; Shigeru Motomura; Toshihiro Suda (1059-1067).
Urocortin (Ucn) 1, Ucn2, and Ucn3 have potent effects on appetite and the cardiovascular system. Endogenous Ucns in combination with CRF receptor type 2β may have a physiological role in the cardiovascular system. We previously demonstrated that both Ucn1 and Ucn2 increased IL-6 output levels in A7r5 aortic smooth muscle cells. In the present study, we extended observations on stress or hormone-induced changes in IL-6 gene expression in the cardiovascular system, and determined the effects of glucocorticoids on Ucn-mediated increases in IL-6 mRNA levels, protein levels, and gene transcription activity in A7r5 cells. Ucn1, Ucn2, and Ucn3 all increased IL-6 mRNA levels via CRF receptor type 2. Dexamethasone blocked the ability of Ucn1 to increase IL-6 mRNA and protein levels, while it failed to attenuate the Ucns-mediated changes in cyclic AMP (cAMP)-response element binding protein or extracellular signal-related kinases phosphorylation. Dexamethasone also suppressed Ucn1- or cAMP-stimulated IL-6 gene transcription via a glucocorticoid receptor. Together, these findings demonstrate that glucocorticoids suppress IL-6 gene transcription via Ucn-induced cAMP-dependent pathways in A7r5 cells.
Keywords: Urocortin; Cyclic AMP; Corticotropin-releasing factor receptor; Glucocorticoids; Interleukin-6;

Increased levels of C-type natriuretic peptide in patients with idiopathic left ventricular dysfunction by Silvia Del Ry; Daniela Giannessi; Maristella Maltinti; Concetta Prontera; Annalisa Iervasi; Chiara Colotti; Michele Emdin; Antonio L’Abbate; Danilo Neglia (1068-1073).
C-type natriuretic peptide (CNP) is expressed in the vascular endothelium. It is not known whether CNP is specifically increased in patients with idiopathic left ventricular systolic dysfunction (ILVDys) with or without overt heart failure, and whether in these patients it is related with indicators of myocardial and/or endothelial/microvascular impairment.We determined plasma CNP levels in 51 ILVDys and in 60 controls. We observed a significant increase in patients with (7.0 ± 0.9 pg/ml) or without (6.1 ± 0.53 pg/ml) overt heart failure (p  < 0.001) in respect to controls (2.5 ± 0.12 pg/ml).CNP was significantly correlated with LVEF (p  < 0.001), end-diastolic dimension (p  < 0.05), ANP (p  < 0.001) and BNP (p  < 0.001), interleukin-6 (p  < 0.001), total cholesterol (p  < 0.05), low-density lipoprotein (p  = 0.05), ratio total cholesterol/ high-density lipoprotein (p  = 0.05) and, in a subgroup of patients, with abnormal vasodilating capacity of the coronary microcirculation.In conclusion, CNP is activated in patients with LV dysfunction but without coronary artery disease, independently of the presence of overt heart failure and in tune with the extent of myocardial functional involvement. In these patients CNP is also related with both systemic and coronary indicators of endothelial/microvascular damage.
Keywords: C-type natriuretic peptide (CNP); Natriuretic peptides; Idiopathic left ventricular systolic dysfunction (ILVDys);

Development of photolabile caged analogs of endothelin-1 by S. Bourgault; M. Létourneau; A. Fournier (1074-1082).
Photoactivable caged analogs of endothelin-1 (ET-1) were obtained after derivatization with the photolabile 4,5-dimethoxynitrobenzyl (DMNB) group. This was achieved by the incorporation of N-α-Fmoc caged building blocks of Lys, Asp, Glu and Tyr during the solid phase peptide synthesis step. The C-terminal carboxylic function was also derivatized. However, difficulties were encountered with the introduction of the Asp and Glu photoactivable building blocks. As a matter of fact, formation of an aminosuccinyl derivative, through cyclization of the Asp(ODMNB) residue, and the formation of a pyrrolidone ring from the Glu(ODMNB) residue were highly favored by the electronic properties of the photocleavable function. ET-1 analogs were also tested in the ETA and ETB paradigms and specific pharmacological profiles were obtained for each peptide.
Keywords: Endothelin; Caged; Nitrobenzyl; Photoactivation;

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.
Keywords: Endothelin-2; UVC radiation; Gene expression; Skin;

Expression of adrenomedullin2/intermedin in human brain, heart, and kidney by Ryo Morimoto; Fumitoshi Satoh; Osamu Murakami; Kazuhito Totsune; Takashi Suzuki; Hironobu Sasano; Sadayoshi Ito; Kazuhiro Takahashi (1095-1103).
Adrenomedullin2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. In the present study, we developed a specific radioimmunoassay of human AM2/IMD. Expression of AM2/IMD was studied in the human brain, pituitary, heart and kidney obtained at autopsy by radioimmunoassay and immunocytochemistry. Immunoreactive-AM2/IMD was detected by radioimmunoassay in human brains (range; 0.163–1.495 pmol/g wet weight), pituitaries (4.46 ± 0.689 pmol/g wet weight, mean ± S.E.M, n  = 3), left ventricles of hearts (0.251 ± 0.0321 pmol/g wet weight, n  = 4), kidneys (3.49 ± 1.18 pmol/g wet weight, n  = 5), and plasma obtained at healthy subjects (24.7 ± 1.78 pmol/l, n  = 3). Reverse-phase high performance liquid chromatography showed that immunoreactive-AM2/IMD in human brain, kidney and plasma extracts were eluted in the position of authentic AM2/IMD. Additional peaks eluted earlier were found in the brain tissue and plasma. Immunocytochemistry showed that immunoreactive-AM2/IMD was localized in paraventricular and supraoptic nuclei of hypothalamus, anterior and posterior lobes of pituitary, cardiomyocytes, pericardial adipocytes, vascular endothelial cells of pericardial veins, and vascular smooth muscle cells of coronary arteries and renal arterioles as well as in renal tubular cells. The present study has shown expression of AM2/IMD in various types of cells in the central nervous system and the cardiovascular system, and suggested possible (patho)physiological roles of AM2/IMD in these systems.
Keywords: Adrenomedullin; Intermedin; Radioimmunoassay; Pituitary; Kidney; Heart;

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats by Hirofumi Hashimoto; Susumu Hyodo; Makoto Kawasaki; Minori Shibata; Takeshi Saito; Hitoshi Suzuki; Hiroki Otsubo; Toru Yokoyama; Hiroaki Fujihara; Takashi Higuchi; Yoshio Takei; Yoichi Ueta (1104-1112).
Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM22-52 and calcitonin gene-related peptide (CGRP) antagonist, CGRP8-37 on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM22-52 (27 nmol/rat) and CGRP8-37 (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM22-52  + CGRP8-37 could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.
Keywords: AM/CGRP receptor; c-fos; In situ hybridization histochemistry; Paraventricular nucleus; Supraoptic nucleus;

Central oxytocin enhances antinociception in the rat by Jun Yang; Yu Yang; Jian-Min Chen; Wen-Yan Liu; Cheng-Hai Wang; Bao-Chen Lin (1113-1119).
The study aimed to investigate the effect of oxytocin on antinociception in the rat. The pain threshold was elevated by oxytocin following intraventricular (icv) or intrathecal injection (ith), and reduced by anti-oxytocin serum (icv or ith). But the pain threshold was not altered by intravenous injection (iv) of oxytocin or anti-oxytocin serum. Pain stimulation induced oxytocin concentration decrease in the hypothalamic supraoptic nucleus, and increase in the locus coeruleus, raphe magnus nucleus, caudate nucleus and spinal cord, but no change in the hypothalamic paraventricular nucleus and plasma. The results indicated that central, not peripheral oxytocin could enhance antinociception.
Keywords: Oxytocin; Anti-oxytocin serum; Antinociception; Central nervous system; Peripheral system; Hypothalamic supraoptic nucleus;

Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus by Alice Blackshear; Mihoko Yamamoto; Brenda J. Anderson; Philip V. Holmes; Linda Lundström; Ülo Langel; John K. Robinson (1120-1124).
The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and post-synaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebroventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated regulation of stress-responses, food intake, and Pavlovian conditioning.
Keywords: Anxiety; Feeding; Neuropeptide; Motivation;

Activation of the NPFF2 receptor reduces the inhibitory effect of opioids on the N-type Ca2+ channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and α2-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF2 receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca2+ transient by activation of Gi-coupled receptors was either unaffected (μ-opioid), increased (NPY), reduced (NPFF2) or lost (α2-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the μ-opioid and NPFF2 receptors to the Gi/o proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

Adrenomedullin is a novel adipokine: Adrenomedullin in adipocytes and adipose tissues by Yin Li; Changtao Jiang; Xian Wang; Yan Zhang; Shigeki Shibahara; Kazuhiro Takahashi (1129-1143).
Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. The production and the secretion of AM have been demonstrated in cultured adipocytes and adipose tissues. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and lipopolysaccharide are strong stimulators for AM expression in adipocytes. Furthermore, AM expression in the adipose tissue is increased in obesity, and plasma concentrations of AM are increased in obese subjects. One possible (patho)physiological role of AM secreted by adipose tissue may be actions against complications of the metabolic syndrome characterized by obesity, type 2 diabetic mellitus and hypertension, via its antioxidant and potent vasodilator effects. These findings indicate that AM is a new member of the adipokine family.
Keywords: Adrenomedullin; Adipocytes; Metabolic syndrome; Adipokine;

Role of plant lipid transfer proteins in plant cell physiology—A concise review by André de Oliveira Carvalho; Valdirene Moreira Gomes (1144-1153).
Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.
Keywords: Antimicrobial activity; Antimicrobial peptides; Gene expression; Lipid transfer protein; Plant signaling;