Peptides (v.29, #7)

The key-role of tyrosine 155 in the mechanism of prion transconformation as highlighted by a study of sheep mutant peptides by Gildas Bertho; Guillaume Bouvier; Gaston Hui Bon Hoa; Jean-Pierre Girault (1073-1084).
Prion protein is a strongly conserved and ubiquitous glycoprotein. The conformational conversion of the non-pathogenic cellular prion isoform (PrPC) into a pathogenic scrapie isoform (PrPSc) is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this conversion, helix H1 and its two flanking loops are known to undergo a conformational transition into a β-like structure. In order to understand mechanisms which trigger this transconformation, sheep prion protein synthetic peptides spanning helix 1 and β-strand 2 (residues 142–166) were studied: (1) the N3 peptide, studied earlier, is known to fold into β-hairpin-like conformation in phosphate buffer at neutral pH and to adopt a helix H1 conformation when dissolved in trifluoroethanol/phosphate buffer mixture, (2) The R156A mutant (peptide R15) and (3) the Y155A mutant (peptide Y14) of the N3 peptide are studied by circular dichroism and NMR spectroscopy in this article. Structural characterization of these peptides highlights the key role of tyrosine 155 in the stabilization of the β-hairpin-like conformation of the sheep peptide in phosphate buffer. We propose a model where tyrosine 155 could stabilize the β-hairpin structure by creating a hydrophobic core in phosphate buffer, necessary to initiate the β-type structure formation. In the turn, the side chain ionic interaction, E152-R156 described before, seems to play a minor role relative to the hydrophobic packing, as observed with the R156A mutation (peptide R15). Interestingly, homology at amino acid residue 155 could be responsible for the species barrier in TSE.
Keywords: NMR; Hydrophobic packing; Salt bridges; Prion protein; Fragments 152–156; Solvation;

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: Predicting experimental toxicity by Allison A. Langham; Himanshu Khandelia; Benjamin Schuster; Alan J. Waring; Robert I. Lehrer; Yiannis N. Kaznessis (1085-1093).
The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are β-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.
Keywords: Antimicrobial peptides; Toxicity prediction; Protegrin; Molecular dynamics simulation; Quantitative structure activity relationship (QSAR);

Mussels have diverse groups of cysteine rich, cationic antimicrobial peptides (AMPs) (defensins, mytilins, myticins, and mytimycin) that constitute an important component of their innate immune defence. Despite the identification and characterization of these AMPs in mussels, the underlying genetic mechanisms that maintain high diversity among multiple variants of the myticin-C isoform are poorly understood. Using phylogeny-based models of sequence evolution and several site-by-site frequency spectrum statistical tests for neutrality, herein we report that positive selection has been the major driving force in maintaining high diversity among the allelic-variants of the myticin-C AMP of Mytilus galloprovincialis. The statistical tests rejected the hypothesis that all polymorphism within myticin-C loci is neutral. Although a majority of the codons constrained to purifying selection (rate of amino acid replacement to the silent substitution, ω  < 1), approximately 8% of the codons with ω  ≈ 5.5 are under positive selection (ω  > 1), thus indicating adaptive evolution of certain amino acids. Direct interaction of these peptides with the surrounding pathogens and/or altered/new pathogens in the changing environment is the likely cause of molecular adaptation of certain amino acid sites in myticin-C variants.
Keywords: Mussel; Antimicrobial peptides; Myticin; Molecular evolution; Maximum-likelihood models; Positive selection;

Structure–activity relations of parasin I, a histone H2A-derived antimicrobial peptide by Young Sook Koo; Jung Min Kim; In Yup Park; Byung Jo Yu; Su A Jang; Key-Sun Kim; Chan Bae Park; Ju Hyun Cho; Sun Chang Kim (1102-1108).
The structure–activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic α-helical structure (residues 9–17) flanked by two random coil regions (residues 1–8 and 18–19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2–19)] resulted in loss of antimicrobial activity, but did not affect the α-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R1]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1–17), Pa(1–15)] slightly increased the antimicrobial activity (1–4 μg/ml) without affecting the α-helical content of the peptide. However, further deletion [Pa(1–14)] resulted in nearly complete loss of antimicrobial activity and α-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1–14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2–19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the α-helical structure is necessary for the membrane-permeabilizing activity.
Keywords: Parasin I; Histone H2A; Antimicrobial peptide; Membrane permeabilization;

The synthetic antimicrobial peptide representative of the first 11 N-terminal amino acids of human lactoferrin (hLF 1–11) kills multidrug-resistant Staphylococcus aureus (MRSA). This study displays antimicrobial activity of hLF 1–11, via various routes of administration, against MRSA infections in mice. Radiolabeling hLF 1–11 with technetium-99m (99mTc-hLF 1–11) enables scintigraphic monitoring directly after administration. 99mTc-hLF 1–11 was taken up by the gall bladder, intestines, and kidneys. Most of the radioactivity was captured in the urinary bladder and about 1% of the injected dose accumulated into infected thigh muscles. At 2 or 24 h after either intravenously, subcutaneously, intraperitoneally, or orally injected a single dose of 0.04 mg/kg hLF 1–11 in mice significantly reduced (20–60 times) the number of viable MRSA. In a dose-response setting in immunocompetent mice maximum bactericidal effects (10,000 times reduction) of intravenously injected 99mTc-hLF 1–11 was seen with 40 mg/kg whereas the same dose of orally administered 99mTc-hLF 1–11 induced about approximately 100 times reduction. In conclusion, intravenously and orally administrated 99mTc-hLF 1–11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues.
Keywords: MRSA infection; Lactoferrin peptide; 99mTc-labeling; Routes of administration; Scintigraphy; Antibacterial therapy;

BMAP-28 improves the efficacy of vancomycin in rat models of gram-positive cocci ureteral stent infection by Fiorenza Orlando; Roberto Ghiselli; Oscar Cirioni; Daniele Minardi; Linda Tomasinsig; Federico Mocchegiani; Carmela Silvestri; Barbara Skerlavaj; Alessandra Riva; Giovanni Muzzonigro; Vittorio Saba; Giorgio Scalise; Margherita Zanetti; Andrea Giacometti (1118-1123).
An experimental study was performed to evaluate the efficacy of BMAP-28 alone and in combination with vancomycin in animal models ureteral stent infection due to Enterococcus faecalis and Staphylococcus aureus. Study included a control group without bacterial challenge to evaluate the sterility of surgical procedure, a challenged control group that did not receive any antibiotic prophylaxis and for each bacterial strain three challenged groups that received (a) 10 mg/kg vancomycin intraperitoneally, immediately after stent implantation, (b) BMAP-28-coated ureteral stents where 0.2-cm2 sterile ureteral stents were incubated in 1 mg/l BMAP-28 solution for 30 min immediately before implantation and (c) intraperitoneal vancomycin plus BMAP-28-coated ureteral stent at the above concentrations. Experiments were performed in duplicate. Ureteral stents were explanted at day 5 following implantation and biofilm bacteria enumerated. Our data showed that rats that received intraperitoneal vancomycin showed the lowest bacterial numbers. BMAP-28 combined with vancomycin showed efficacies higher than that of each single compound. These results highlight the potential usefulness of this combination in preventing ureteral stent-associated in gram-positive infections.
Keywords: Ureteral stent; Biofilm; Cathelicidins; Animal model; Gram-positive cocci;

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum–corpus allatum: A case study with beetles and moths by Gerd Gäde; Heather G. Marco; Petr Šimek; Neil Audsley; Kevin D. Clark; Robert J. Weaver (1124-1139).
This mass spectrometric study confines itself to peptide masses in the range of 500–1500 Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a γ-PGN-(=γ-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.
Keywords: Insects; Neuropeptides; Adipokinetic peptides; Genome prediction; MALDI-TOF MS; LC-ESI MS; Tribolium castaneum; Tribolium brevicornis; Bombyx mori; Spodoptera species; Lacanobia oleracea; Mamestra brassicae;

Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide by Julie Clark; Maja Milakovic; Amanda Cull; Markus K. Klose; A. Joffre Mercier (1140-1149).
DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10−8  M. The increase in tonus persisted in the presence of 7 × 10−3  M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.
Keywords: FMRFamide; Postsynaptic modulation; Calcium channel;

Pharmacologic study of C-terminal fragments of frog skin calcitonin gene-related peptide by Ali Ladram; Isabelle Besné; Lionel Breton; Olivier de Lacharrière; Pierre Nicolas; Mohamed Amiche (1150-1156).
The calcitonin gene-related peptide from the skin of the frog Phyllomedusa bicolor (pbCGRP) is a 37-residue neuropeptide that differs from human α CGRP (hαCGRP) at 16 positions. The affinities of the C-terminal fragments of pbCGRP and hαCGRP were evaluated in SK-N-MC cells: pbCGRP8-37 (K i  = 0.2 nM) and pbCGRP27-37 (K i  = 95 nM) were, respectively, 3 times and 20 times more potent than the human fragments hαCGRP8-37 and hαCGRP27-37. Their antagonistic potencies were measured in SK-N-MC and Col 29 cells, and the rat vas deferens. pbCGRP8-37 inhibited the hαCGRP-stimulated production of cAMP by SK-N-MC and Col 29 cells 3 to 4 times more strongly than hαCGRP8-37. Thus pbCGRP8-37 is the most potent CGRP-1 competitive antagonist of all the natural sequences reported to date. pbCGRP27-37 was also as potent as [D31, A34, F35] hαCGRP27-37, a prototypic antagonist analog derived from structure–activity relationship studies of hαCGRP8-37.
Keywords: Amphibian; Phyllomedusa; pbCGRP; Antagonist;

Bombesin: A possible role in wound repair by A. Baroni; B. Perfetto; N. Canozo; A. Braca; E. Farina; A. Melito; S. De Maria; M. Cartenì (1157-1166).
During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFβ, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFβ, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.
Keywords: HaCat; Bombesin; Wound healing; COX-2;

The motilin receptor (MTLR) is an important therapeutic target for the treatment of hypomotility disorders but desensitization may limit its clinical utility. The aim of this study was to investigate the role of the C-terminal tail of the MTLR in the desensitization, phosphorylation and internalization process. Three MTLR mutants, C-terminally truncated from amino acid 412 till 384 (MTLRΔ385), 374 (MTLRΔ375) or 368 (MTLRΔ369), were constructed and C-terminally tagged with an EGFP and stably expressed in CHO cells co-expressing the Ca2+ indicator apoaequorin. Activity and desensitization were studied by measuring changes in motilin-induced luminescent Ca2+ rises. Receptor phosphorylation was investigated by immunoprecipitation and MTLR-EGFP internalization was visualized by fluorescence microscopy. Truncation only reduced MTLR affinity and the efficacy to induce Ca2+ luminescent responses of the MTLRΔ375-EGFP mutant. Furthermore, the region between amino acid 375 and 368 seems to be important for proper cell surface expression of the MTLR since receptors of the MTLRΔ369-EGFP mutant but not of the other mutants were found intracellularly in vesicles. Truncation of the receptor till amino acid 384 or 374 did neither affect desensitization nor internalization. In contrast phosphorylation of the MTLRΔ385-EGFP mutant was reduced by 80% but was not affected in the MTLRΔ375-EGFP mutant. In conclusion, MTLR desensitization and internalization is not dependent on the presence of the C-terminal tail. Truncation favors internalization via either phosphorylation-independent pathways or via phosphorylation of alternative sites in the receptor.
Keywords: Motilin receptor; Desensitization; Internalization; Phosphorylation; C-terminal truncation; Motilin receptor mutants; Ca2+ luminescence;

The decapeptide CMS001 enhances swimming endurance in mice by Li Wang; Hua-Li Zhang; Rong Lu; Yan-Jiao Zhou; Rui Ma; Jun-Qiang Lv; Xiao-Lei Li; Li-Juan Chen; Zhi Yao (1176-1182).
Now peptides achieve distinct advantages over protein in biological application because of its quick and easy absorption, low power, and high activity. Some bioactive peptides had been developed to be used in the management of exercise-related disorders. In this study, we investigated whether the decapeptide CMS001 (Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe) isolated from pig spleen had anti-fatigue effects. Male Balb/c mice were administered CMS001 (20 μg/(kg d)−1 or 5 μg/(kg d)−1 for 30 d, intraperitoneal injections) and tested in an exhaustive swim time task. In order to examine the mechanisms of CMS001 anti-fatigue effects, we analyzed liver glycogen stores, blood urea nitrogen (BUN) levels, lactic acid levels, ultrastructural integrity, and levels of both a free radical metabolite and an anti-oxidant enzyme. CMS001 treatment prolonged exhaustive swim time, increased liver glycogen levels, reduced BUN levels, and decreased accumulation of lactic acid in the blood, relative to mice injected with only saline. Examination of the ultrastructure of mitochondria and sarcoplasmic reticulum in skeletal and cardiac muscle of CMS001-treated and control mice revealed that CMS001 can reduce the damage to cardiac and skeletal muscle caused by an exhaustive swim challenge, such that the structure of most tissue specimens were normal in the peptide-treated group. Furthermore the free radical analysis after acute exercise indicated that CMS001 treatment decreased malondialdehyde (MDA) and increased superoxide dismutase (SOD) levels. The present findings indicate that the spleen-derived peptide CMS001 has anti-fatigue effects in mice, and further suggest that the mechanism may involve reduction of tissue damaging free radicals in muscle tissues.
Keywords: Small molecular peptide; Exercise-induced fatigue; Exhaustive swimming; Free radical;

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice by Yi-qing Wang; Jia Guo; Sheng-bin Wang; Quan Fang; Feng He; Rui Wang (1183-1190).
The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5 nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe1]NC(1-13)NH2 (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.
Keywords: Opioid; Nociceptin/orphanin FQ (N/OFQ); Morphine; Neuropeptide FF (NPFF); RF9; Hypothermia; Mice;

Substance P (SP) is known to be involved in processes related to learning and memory, fear, anxiety and stress. SP and NK1 receptors are localized in the hippocampus, a brain structure involved in learning and memory as well as emotional processes. As there is evidence for differential functions of the ventral (VH) and dorsal (DH) hippocampus in a variety of behaviors, we here evaluated the effects of injections of SP into the VH and DH in rats submitted to the elevated plus-maze (EPM) and open field (OF) tests. The results obtained showed that infusions of 100 and 1000 ng of SP into the DH, but not VH, increased open arm activity in the EPM and in the central zone of the OF, indicative of anxiolytic-like action. These effects were observed in the absence of significant changes in general motor activity. In an additional experiment to examine whether these effects of SP are mediated by local serotoninergic mechanisms, extracellular concentrations of this monoamine were assessed by use of in vivo microdialysis. Infusions of SP into the DH did not influence the extracellular concentration of serotonin. These data indicate that neurokinins in the DH, but not VH, are involved in mechanisms associated with anxiety and that the mediation of SP in anxiety-related behaviors is independent of local serotonergic mechanisms.
Keywords: Anxiety; Elevated plus-maze; Substance P; Dorsal hippocampus; Ventral hippocampus; Rat;

Detection of stable N-terminal protachykinin A immunoreactivity in human plasma and cerebrospinal fluid by Andrea Ernst; Jennifer Suhr; Josef Köhrle; Andreas Bergmann (1201-1206).
Substance P (SP) is a neuropeptide that is released from sensory nerves and several types of immune cells. It is involved in the transmission of pain and has a number of pro-inflammatory effects. Like other neuropeptides, SP is derived from a large precursor peptide, protachykinin A (PTA). Alternative splicing results in the production of four distinct PTA molecules that all contain the sequence of SP and a common N-terminal region consisting of 37 amino acids. We have developed a sandwich immunoassay using antibodies against the N-terminal part of PTA. Here we demonstrate that N-terminal PTA immunoreactivity is present in human circulation and cerebrospinal fluid (CSF). The concentration was about 90 times higher in CSF than in EDTA-plasma. Analytical reversed phase HPLC revealed that NT-PTA 1-37 is the main immunoreactivity in human circulation and CSF. Moreover, compared to the low in vitro stability of SP of less than 12 min, NT-PTA immunoreactivity is absolutely stable in EDTA-plasma and CSF for more than 48 h. As NT-PTA 1-37 is produced in stoichiometric amounts and is theoretically co-released with SP, we suggest the measurement of NT-PTA immunoreactivity as surrogate molecule for the release of bioactive SP.
Keywords: Substance P; Tachykinin A; Protachykinin A; CSF; Neuropeptide; NT-PTA; Immunoassay;

Different response of ANP secretion to adrenoceptor stimulation in renal hypertensive rat atria by Kuichang Yuan; Kyoung-Suk Rhee; Woo Hyun Park; Soo Wan Kim; Suhn Hee Kim (1207-1215).
Sympathetic nervous system and atrial natriuretic peptide (ANP) system play fundamental roles in the regulation of cardiovascular functions. Overactivity of sympathetic nervous system can lead into cardiovascular diseases such as heart failure and hypertension. The present study aimed to define which adrenergic receptors (ARs) affect atrial contractility and ANP release and to determine their modification in renal hypertensive rat atria. An α1-AR agonist, cirazoline increased ANP release with positive inotropism. These α1-AR agonist-mediated responses were attenuated by the α1A-AR antagonist, but not by the α1B- or α1D-AR antagonist. An α2-AR agonist, guanabenz and clonidine increased ANP release with negative inotropism and decreased cAMP level. The order of potency for the increased ANP release was cirazoline ≫ phenylephrine = guanabenz ≫ clonidine. In contrast, a β-AR agonist, isoproterenol decreased ANP release with positive inotropism and these responses were blocked by the β1-AR antagonist but not by the β2-AR antagonist. The increased cAMP level by isoproterenol was suppressed by pretreatment with both β1- and β2-AR antagonists. In renal hypertensive rat atria, the effects of isoproterenol on atrial contractility, ANP release, and cAMP level were attenuated whereas the effect of cirazoline on ANP release was unaltered. Atrial β1-AR mRNA level but not α1A-AR mRNA level was decreased in renal hypertensive rats. These findings suggest that α1A- and β1-AR oppositely regulate atrial ANP release and that atrial β1-AR expression/function is impaired in renal hypertensive rats.
Keywords: ANP; Atrium; Rat; Adrenoceptor; Hypertension;

Oxytocin alleviates hepatic ischemia–reperfusion injury in rats by Fikret Düşünceli; Sevgin Ö. İşeri; Feriha Ercan; Nursal Gedik; Cumhur Yeğen; Berrak Ç. Yeğen (1216-1222).
Various mechanisms have been proposed for the pathogenesis of postischemic hepatic injury, including the generation of reactive oxygen metabolites. Oxytocin (OT) possesses antisecretory, antiulcer effects, facilitates wound healing and has anti-inflammatory properties. Hepatic ischemia–reperfusion (I/R)-injury was induced by inflow occlusion to median and left liver lobes (∼70%) for 30 min of ischemia followed by 1 h reperfusion in female Sprague–Dawley rats under anesthesia. I/R group (n  = 8) was administered intraperitoneally either OT (500 μg/kg) or saline at 24 and 12 h before I/R and immediately before reperfusion. Sham-operated group that underwent laparotomy without hepatic ischemia served as the control. Rats were decapitated at the end of reperfusion period. Hepatic samples were obtained for the measurement of myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH) and collagen levels and histopathological analysis. Tumor necrosis factor-alfa (TNF-α) and transaminases (SGOT, SGPT) were assayed in serum samples. I/R injury caused significant increases in hepatic microscopic damage scores, MPO activity, collagen levels, transaminase, serum TNF-α levels. Oxytocin treatment significantly reversed the I/R-induced elevations in serum transaminase and TNF-α levels and in hepatic MPO and collagen levels, and reduced the hepatic damage scores. OT treatment had tendency to abolish I/R-induced increase in MDA levels, while GSH levels were not altered. These results suggest that OT has a protective role in hepatic I/R injury and its protective effect in the liver appears to be dependent on its inhibitory effect on neutrophil infiltration.
Keywords: Liver; Ischemia/reperfusion injury; TNF-α; Oxytocin; Neutrophils;

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock by Rongqian Wu; Weifeng Dong; Xiaoling Qiang; Youxin Ji; Tianpen Cui; Juntao Yang; Mian Zhou; Steven Blau; Corrado P. Marini; Thanjavur S. Ravikumar; Ping Wang (1223-1230).
We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague–Dawley rats were bled to and maintained at an MAP of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 μg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4 h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4 h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-α and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4 h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.
Keywords: Hemorrhage; Adrenomedullin; Adrenomedullin binding protein-1; Survival;

Ghrelin improves burn-induced multiple organ injury by depressing neutrophil infiltration and the release of pro-inflammatory cytokines by Özer Şehirli; Emre Şener; Göksel Şener; Şule Çetinel; Can Erzik; Berrak Ç. Yeğen (1231-1240).
Mechanisms of burn-induced skin and remote organ injury involve oxidant generation and the release of pro-inflammatory cytokines. In this study the possible antioxidant and anti-inflammatory effects of ghrelin were evaluated in a rat model of thermal trauma. Wistar albino rats were exposed to 90 °C bath for 10 s to induce thermal trauma. Ghrelin, was administered subcutaneously (10 ng/kg/day) after the burn injury and repeated twice daily. Rats were decapitated at 6 h and 48 h after burn injury and blood was collected for the analysis of pro-inflammatory cytokines (TNF-α and IL-1β), lactate dehydrogenase (LDH) activity and antioxidant capacity (AOC). In skin, lung and stomach tissue samples malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na+–K+-ATPase activity were measured in addition to the histological analysis. DNA fragmentation ratio in the gastric mucosa was also evaluated. Burn injury caused significant increase in both cytokine levels, and LDH activity, while plasma AOC was found to be depleted after thermal trauma. On the other hand, in tissue samples the raised MDA levels, MPO activity and reduced GSH levels, Na+–K+-ATPase activity due to burn injury were found at control levels in ghrelin-treated groups, while DNA fragmentation in the gastric tissue was also reduced. According to the findings of the present study, ghrelin possesses a neutrophil-dependent anti-inflammatory effect that prevents burn-induced damage in skin and remote organs and protects against oxidative organ damage.
Keywords: Thermal trauma; Ghrelin; Cytokine; Lipid peroxidation; Myeloperoxidase;

Ghrelin infused into the portal vein inhibits glucose-stimulated insulin secretion in Wistar rats by Can Cui; Hiroshi Ohnuma; Makoto Daimon; Shinji Susa; Hiroshi Yamaguchi; Wataru Kameda; Yumi Jimbu; Toshihide Oizumi; Takeo Kato (1241-1246).
Although accumulating evidence has shown crucial roles of ghrelin and insulin in food intake and energy metabolism, the exact relationship between these hormones remains unclear. In this study, we determined the in vivo effect of ghrelin on insulin secretion. We demonstrated that ghrelin inhibited the glucose-stimulated release of insulin when infused into the portal vein of Wistar rats. However, ghrelin infusion into the femoral vein did not induce such an inhibitory effect. Hepatic vagotomy or coinfusion with atropine methyl bromide diminished the inhibitory effect of ghrelin on glucose-stimulated insulin secretion. In conclusion, ghrelin exerts an inhibitory effect on glucose-stimulated insulin secretion via the hepatic portal system and the vagus nerve. The decrease in ghrelin level after a meal is important for the occurrence of the incretin effect in rats.
Keywords: Ghrelin; Insulin; Glucose tolerance test; Hepatic portal system; Vagus nerve;

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats by Zhi-Fu Guo; An-Jing Ren; Xing Zheng; Yong-Wen Qin; Fang Cheng; Jing Zhang; Hong Wu; Wen-Jun Yuan; Lin Zou (1247-1254).
Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2 h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48 h fasted rats and 48 h fasted rats refed 2 h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.
Keywords: Ghrelin; Obestatin; Food intake; Food composition; Rat;

Ghrelin, des-acyl ghrelin and obestatin: Three pieces of the same puzzle by João-Bruno Soares; Adelino F. Leite-Moreira (1255-1270).
The major active product of ghrelin gene is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, called simply ghrelin. Ghrelin has a multiplicity of physiological functions, affecting GH release, food intake, energy and glucose homeostasis, gastrointestinal, cardiovascular, pulmonary and immune function, cell proliferation and differentiation and bone physiology. Nevertheless, recent developments have shown that ghrelin gene can generate various bioactive molecules besides ghrelin, mainly des-acyl ghrelin and obestatin, obtained from alternative splicing or from extensive post-translational modification. Although their receptors have not yet been identified, they have already proven to be active, having intriguingly subtle but opposite physiological actions to ghrelin. This suggests the existence of a novel endocrine system with multiple effector elements which not only may have opposite actions but may regulate the action of each other. In this review, we summarize the steps which lead to the production of the different ghrelin gene products and examine the most significant differences between them in terms of structure and actions.
Keywords: Ghrelin; Des-acyl ghrelin; Obestatin; GHS-R1a; Ghrelin gene;