Peptides (v.30, #9)

Plant defensin corn 1 (Pdc1) gene was amplified from corn genomic DNA with the primers designed from a corn EST sequence homologous to a barley defensin gene. The cloned gene contains two exons and an intron. The deduced 9 KDa PDC1 peptide has a sequence that is identical to corn γ2-zeathionin and has the typical features of a plant defensin, including a signal sequence of 35 amino acids, followed by a characteristic defensin domain of 47 amino acids containing 8 cysteines. The defensin protein was expressed from the cloned cDNA introduced into two different expression systems; prokaryotic, Escherichia coli and eukaryotic, yeast (Pichia pastoris). The PDC1 protein was purified with a nickel resin column and was tested for its antifungal activities using the pathogen Fusarium graminearum. The protein expressed in both E. coli and P. pastoris had antifungal activity, however the protein expressed in P. pastoris was more efficient in inhibiting growth of F. graminearum. FTIR analysis of PDC1 protein expressed in the two expression systems showed that expression in P. pastoris gave a product with more β-sheets and less random unordered structure than when it was expressed in E. coli. In addition, removal of the His-tag used for purification increased the fungicidal activity of the PDC1 protein. The data presented here suggest that the defensin PDC1 peptide of corn could be effectively used to restrict the disease caused by F. graminearum.
Keywords: Antifungal activity; Defensin; Fusarium graminearum; Protein expression; Zea mays;

Comparative proteomic analysis of bacterial wilt susceptible and resistant tomato cultivars by Amber Afroz; Muhammad Rashid Khan; Nagib Ahsan; Setsuko Komatsu (1600-1607).
To investigate the molecular mechanisms of bacterial resistance in susceptible and resistant cultivars of tomato, a proteomic approach was adopted. Four cultivars of tomato were selected on the basis of their response to bacterial (Pseudomonas solanacearum) inoculation wherein cultivar Roma and Riogarande, and cultivar Pusa Ruby and Pant Bahr were considered as resistant and susceptible cultivars, respectively. Proteins were extracted from leaves of 3-week-old seedlings of the four cultivars and separated by 2-DE. A total of nine proteins were found to be differentially expressed between the susceptible and resistant cultivars. Amino acid sequences of these proteins were determined with a protein sequencer. The identified proteins belongs to the categories of energy, protein destination and storage, and defense. Of these proteins, a 60 kDa chaperonin and an apical membrane antigen were significantly upregulated in resistant cultivars compared with susceptible cultivars. Application of jasmonic acid and salicylic acid resulted in significant changes in levels of apical membrane antigen and protein disulfide-isomerase. Taken together, these results suggest that apical membrane antigen might be involved in bacterial resistance process through salicylic acid induced defense mechanism signaling in tomato plants.
Keywords: Bacterial wilt; Jasmonic acid; Proteome; Salicylic acid; Tomato;

KKKKPLFGLFFGLF: A cationic peptide designed to exert antibacterial activity by Emilie Duval; Céline Zatylny; Mathieu Laurencin; Michèle Baudy-Floc’h; Joël Henry (1608-1612).
With 14 residues organized as two domains linked by a single proline, the de novo peptide called K4 was designed, using Antimicrobial Peptide Database, to exert antibacterial activity. The N-terminal domain is composed of four lysines enhancing membrane interactions, and the C-terminal domain is putatively folded into a hydrophobic α-helix. Following the synthesis, the purification and the structural checking, antibacterial assays revealed a strong activity against gram-positive and gram-negative bacteria including human pathogenic bacteria such as Staphylococcus aureus and some marine bacteria of the genus Vibrio. Scanning electron microscopy of Escherichia coli confirmed that K4 lyses bacterial cells. The cytotoxicity was tested against rabbit erythrocytes and chinese hamster ovary cells (CHO-K1). These tests revealed that K4 is non-toxic to mammalian cells for bacteriolytic concentrations. The peptide K4 could be a valuable candidate for future therapeutic applications.
Keywords: Peptide design; Cationic peptide; α-helix; Antibacterial peptide; Vibrio; Staphylococcus aureus; Cytotoxicity; Cell specificity;

Three-dimensional structure of the two-peptide bacteriocin plantaricin JK by Per Rogne; Christofer Haugen; Gunnar Fimland; Jon Nissen-Meyer; Per Eugen Kristiansen (1613-1621).
The three-dimensional structures of the two peptides, PlnJ and PlnK, that constitutes the two-peptide bacteriocin plantaricin JK have been solved in water/TFE and water/DPC-micellar solutions using nuclear magnetic resonance (NMR) spectroscopy. PlnJ, a 25 residue peptide, has an N-terminal amphiphilic α-helix between Trp-3 and Tyr-15. The 32 residues long PlnK forms a central amphiphilic α-helix between Gly-9 and Leu-24. Measurements of the effect on anti-microbial activity of single glycine replacements in PlnJ and PlnK show that Gly-13 and Gly-17 in both peptides are very sensitive, giving more than a 100-fold reduction in activity when large residues replace glycine. In variants where other glycine residues, Gly-20 in PlnJ and Gly-7, Gly-9, Gly-24 and Gly-25 in PlnK, were replaced, the activity was reduced less than 10-fold. It is proposed that the detrimental effect on activity when exchanging Gly-13 and Gly-17 in PlnJ and PlnK is a result of reduced ability of the two peptides to interact through the GxxxG-motifs constituting Gly-13 and Gly-17.
Keywords: Plantaricin JK; Bacteriocins; Anti-microbial peptides; Peptide structure; Lactic acid bacteria; NMR spectroscopy;

In vitro bactericidal activity of the N-terminal fragment of the frog peptide esculentin-1b (Esc 1–18) in combination with conventional antibiotics against Stenotrophomonas maltophilia by Giuseppantonio Maisetta; Maria Luisa Mangoni; Semih Esin; Giuseppe Pichierri; Anna Lisa Capria; Franca Lisa Brancatisano; Mariagrazia Di Luca; Simona Barnini; Donatella Barra; Mario Campa; Giovanna Batoni (1622-1626).
In this study the bactericidal effect of the N-terminal fragment of the frog skin peptide esculentin-1b [Esc(1–18)] in combination with clinically used antimicrobial agents was evaluated against Stenotrophomonas maltophilia, either in standard conditions (phosphate buffer) or in the presence of human serum. A synergistic bactericidal effect was observed after a 24 h incubation when combinations of Esc(1–18) and amikacin or colistin were used against clinical strains of S. maltophilia with or without resistance to these antibiotics, both in buffer and in the presence of serum. An indifferent effect was observed when the peptide was combined with levofloxacin or ceftazidime. A synergistic effect was also observed at earlier time points when the peptide was used in combination with colistin. Sequential exposure of bacterial cells to Esc(1–18) and amikacin or colistin, or vice versa, indicated that while Esc(1–18) and colistin cooperated in enhancing the bactericidal effect of their combination, when Esc(1–18) was combined with amikacin, the peptide had a major role in initiating the bactericidal effect, while amikacin was required for the subsequent effector phase. Altogether, the results obtained indicate that exposure of S. maltophilia to sub-bactericidal concentrations of Esc(1–18) increases its susceptibility to amikacin or colistin and may also render resistant strains susceptible to these antibiotics.
Keywords: Antimicrobial peptides; Stenotrophomonas maltophilia; Synergism; Esculentin;

In vitro antimicrobial activity of alpha-melanocyte stimulating hormone against major human pathogen Staphylococcus aureus by Madhuri; Tahsina Shireen; S.K. Venugopal; Dipankar Ghosh; Ravisekhar Gadepalli; Benu Dhawan; Kasturi Mukhopadhyay (1627-1635).
Alpha-melanocyte stimulating hormone (α-MSH) is an endogenous anti-inflammatory peptide reported to possess antimicrobial properties, however their role as antibacterial peptides is yet to be established. In the present study, we examined in vitro antibacterial activity of α-MSH against S. aureus strain ISP479C and several methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains. Antibacterial activity was examined by varying several parameters, viz., bacterial cell densities, growth phase, pH, salt concentration, and temperature. Antibacterial activity was also examined in complex biomatrices of rat whole blood, plasma and serum as well as in biofilm form of bacteria. Our results showed that α-MSH possessed significant and rapid antibacterial activity against all the studied strains including MRSA (84% strains were killed on exposure to 12 μM of α-MSH for 2 h). pH change from 7.4 to 4 increased α-MSH staphylocidal activity against ISP479C by 21%. Antibacterial activity of α-MSH was dependent on bacterial cell density and independent of growth phase. Moreover, antimicrobial activity was retained when α-MSH was placed into whole blood, plasma, and serum. Most importantly, α-MSH exhibited antibacterial activity against staphylococcal biofilms. Multiple membrane permeabilization assays suggested that membrane damage was, at least in part, a major mechanism of staphylocidal activity of α-MSH. Collectively the above findings suggest that α-MSH could be a promising candidate of a novel class of antimicrobial agents.
Keywords: α-MSH; Staphylococcus aureus; Antimicrobial peptides; Mechanisms of action; Biofilm;

As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48–96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.
Keywords: Hepcidin; Tumor cell inhibition; Lytic peptide;

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162 by Andrea Treszl; Andrew V. Schally; Stephan Seitz; Luca Szalontay; Ferenc G. Rick; Karoly Szepeshazi; Gabor Halmos (1643-1650).
Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5–100 μM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 μmol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p  < 0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.
Keywords: Cytotoxic somatostatin analog AN-162; Non-small cell lung cancer; Somatostatin receptors; H460 xenograft; H1299 xenograft;

Proteome analysis of tobacco leaves under salt stress by Roya Razavizadeh; Ali Akbar Ehsanpour; Nagib Ahsan; Setsuko Komatsu (1651-1659).
The mechanisms responsible for the effects of salt stress on tobacco plants were examined by means of proteomic analysis. Tobacco plants were exposed to 0, 150, 250, 300, or 400 mM NaCl. At 150 mM NaCl or above, the plants showed a reduction in fresh weight and an increase in proline levels. Proteins extracted from the leaves of tobacco plants exposed to 150 mM NaCl were separated by 2-DE. Of 205 protein spots that were detected reproducibly in each gel, 18 were differentially expressed under NaCl treatment. Up-regulated proteins belonged to the photosynthesis category, whereas down-regulated proteins correspond to defense-related functions. Dose- and time-dependent studies showed that a stromal 70-kDa heat shock-related protein was markedly down-regulated by NaCl. Thus, down-regulation of the stromal 70-kDa heat shock protein in response to salt stress is likely the cause of failure to protect cells against salt stress of tobacco plants.
Keywords: Proline; Proteome; Salt stress; Tobacco;

Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins by Mingming Ma; Theresa M. Szabo; Chenxi Jia; Eve Marder; Lingjun Li (1660-1668).
The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10−6  M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7 Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.
Keywords: Allatostatin; Crustaceans; Neuromodulation; Stomatogastric nervous system; Neuropeptides; Peptide sequencing;

Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of florescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.
Keywords: Cell-penetrating peptide; Cellular internalization; Polyarginine; Protein transduction domain; Fluorescence resonance energy transfer;

Arginine vasopressin in hypothalamic paraventricular nucleus is transferred to the nucleus raphe magnus to participate in pain modulation by Jun Yang; Huifeng Yuan; Wenyan Liu; Chaoyou Song; Hongtao Xu; Gen Wang; Cai Song; Na Ni; Daiwei Yang; Baocheng Lin (1679-1682).
Hypothalamic paraventricular nucleus (PVN) is one of the main sources of arginine vasopressin (AVP) synthesis and secretion. AVP is the most important bioactive substance in PVN regulating pain process. Our pervious study has pointed that pain stimulation induced AVP increase in the nucleus raphe magnus (NRM), which plays a role in pain modulation. The present study was designed to investigate the source of AVP in the rat NRM during pain process using the methods of nucleus push–pull perfusion and radioimmunoassay. The results showed that pain stimulation increased the AVP concentration in the NRM perfusion liquid, PVN cauterization inhibited the role that pain stimulation induced the increase of AVP concentration in the NRM perfusion liquid, and PVN microinjection of l-glutamate sodium, which excited the PVN neurons, could increase the AVP concentration in the NRM perfusion liquid. The data suggested that AVP in the PVN might be transferred to the NRM to participate in pain modulation.
Keywords: Arginine vasopressin; Pain modulation; Hypothalamic paraventricular nucleus; Nucleus raphe magnus;

GPR103 is one of the orphan G protein-coupled receptors. Recently, an endogenous ligand for GPR103, 26RFa, was identified. Many 26RFa binding sites have been observed in various nuclei of the brain involved in the processing of pain such as the parafascicular thalamic nucleus, the locus coeruleus, the dorsal raphe nucleus, and the parabrachial nucleus. In the present study, the effects of intracerebroventricular injection of 26RFa were tested in the rat. Intracerebroventricular injection of 26RFa significantly decreased the number of both phase 1 and phase 2 agitation behaviors induced by paw formalin injection. This analgesic effect of 26RFa on the phase 1 response, but not phase 2 response, was antagonized by BIBP3226, a mixed antagonist of neuropeptide Y Y1 and neuropeptide FF receptors. Intracerebroventricular injection of 26RFa has no effect in the 52.5 °C hot plate test. Intracerebroventricular injection of 26RFa had no effect on the expression of Fos-like immunoreactivity induced by paw formalin injection in the superficial layers of the spinal dorsal horn. These data suggest that (1) 26RFa modulates nociceptive transmission at the supraspinal site during a formalin test, (2) the mechanism 26RFa uses to produce an analgesic effect on the phase 1 response is different from that on the phase 2 response, and (3) intracerebroventricularly injected 26RFa dose not directly inhibit the nociceptive input to the spinal cord.
Keywords: GPR103; Hot plate test; Fos; Inflammtory pain; Supraspinal;

Intrathecal substance P augments morphine-induced antinociception: Possible relevance in the production of substance P N-terminal fragments by Takaaki Komatsu; Mika Sasaki; Kengo Sanai; Hikari Kuwahata; Chikai Sakurada; Minoru Tsuzuki; Yohko Iwata; Shinobu Sakurada; Tsukasa Sakurada (1689-1696).
The present study sought to examine the mechanism of substance P to modulate the antinociceptive action of intrathecal (i.t.) morphine in paw-licking/biting response evoked by subcutaneous injection of capsaicin into the plantar surface of the hindpaw in mice. The i.t. injection of morphine inhibited capsaicin-induced licking/biting response in a dose-dependent manner. Substance P (25 and 50 pmol) injected i.t. alone did not alter capsaicin-induced nociception, whereas substance P at a higher dose of 100 pmol significantly reduced the capsaicin response. Western blots showed the constitutive expression of endopeptidase-24.11 in the dorsal and ventral parts of lumbar spinal cord of mice. The N-terminal fragment of substance P (1–7), which is known as a major product of substance P by endopeptidase-24.11, was more effective than substance P on capsaicin-induced nociception. Combination treatment with substance P (50 pmol) and morphine at a subthreshold dose enhanced the antinociceptive effect of morphine. The enhanced effect of the combination of substance P with morphine was reduced significantly by co-administration of phosphoramidon, an inhibitor of endopeptidase-24.11. Administration of d-isomer of substance P (1–7), [d-Pro2, d-Phe7]substance P (1–7), an inhibitor of [3H] substance P (1–7) binding, or antisera against substance P (1–7) reversed the enhanced antinociceptive effect by co-administration of substance P and morphine. Taken together these data suggest that morphine-induced antinociception may be enhanced through substance P (1–7) formed by the enzymatic degradation of i.t. injected substance P in the spinal cord.
Keywords: Morphine; Substance P; N-terminal fragments of substance P; Capsaicin-induced nociceptive response; Antinociception; Intrathecal injection;

Previously, we have synthesized a series of endomorphin-2 (EM-2) analogs by the substitution of C-terminal amide group. In the present study, to further our knowledge of the influence of C-terminal esterified modification on the pharmacological activities, we investigated the in vitro and in vivo opioid activities of C-terminal esterified EM-2 analogs 13. Our results showed that the ED50 values on contractions of the longitudinal muscle of distal colon induced by analogs 13 were about 1.5-fold higher, 2- and 8-fold lower than EM-2, respectively. In addition, intravenous (i.v.) injections of analogs 1 and 2 dose-dependently decreased the system arterial pressure (SAP) and heart rate (HR) in anesthetized rats, but the degree of the hypotension and bradycardia was significantly smaller relative to the parent. Moreover, analog 3 was almost ineffective. Nevertheless, all these analogs produced potent antinociception in the tail-flick test after intracerebroventricular (i.c.v.) injection, and this antinociception was inhibited by naloxone, indicating an opioid mechanism. In summary, these results gave the evidence that the conversion of C-terminal amide to esterified modification may play an important role in the regulation of opioid affinities and pharmacological activities.
Keywords: Endomorphin-2; C-terminal esterified analogs; Colonic contraction; Cardiovascular response; Antinociception;

Plasma nociceptin/orphanin FQ levels rise after spontaneous episodes of angina, but not during induced myocardial ischemia by Fiorella Fontana; Pasquale Bernardi; Carmine Pizzi; Santi Spampinato; Andrea Bedini; Emilio Merlo Pich (1705-1709).
The aim of our study was to evaluate the effects of repeated episodes of angina and induced myocardial ischemia on plasma nociceptin/orphanin FQ (N/OFQ) levels. Patients with unstable angina (23 with new onset severe angina or accelerated angina and 18 with subacute angina at rest) who had had repeated spontaneous episodes of chest pain in the last week before the study underwent myocardial perfusion single-photon emission computed tomography using adenosine infusion. Twenty subjects without clinical symptoms of angina matched for age, sex and cardiac risk factors served as a control group. N/OFQ levels were significantly (P  < 0.01) higher in the patients (15.2 ± 2.1 pg/ml) than in the control group (8.5 ± 2.6 pg/ml). Blood pressure and heart rate did not significantly differ. All patients showed transient adenosine infusion myocardial ischemia that did not induce chest pain or significantly modify plasma N/OFQ levels or hemodynamic parameters. Our findings show that unstable angina is associated with a significant increase in circulating N/OFQ levels unrelated to intervening transient myocardial ischemia or hemodynamic changes. This increase is probably related to the chest pain repeatedly occurring in the course of coronary artery disease, but absent during transient adenosine-induced myocardial ischemia.
Keywords: Nociceptin/orphanin FQ; Unstable angina; Myocardial scintigraphy; Chest pain;

Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure by Silvia Del Ry; Manuela Cabiati; Vincenzo Lionetti; Anca Simioniuc; Chiara Caselli; Tommaso Prescimone; Michele Emdin; Daniela Giannessi (1710-1713).
High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n  = 8) and without (control, n  = 6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65 ± 0.11 vs. 0.35 ± 0.04, p  = 0.02), but not in the OS (BNP/GAPDH: 0.36 ± 0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089 ± 0.036, PS 0.289 ± 0.23, OS 0.54 ± 0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69 ± 0.13; PS = 1.56 ± 0.19; OS = 1.70 ± 0.42 pg/mg protein; p  = 0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.
Keywords: C-type natriuretic peptide; Natriuretic peptides; Natriuretic peptide receptors; Chronic heart failure;

Acute modulation of myocardial function by angiotensin 1–7 by Paulo Castro-Chaves; Mariana Pintalhao; Ricardo Fontes-Carvalho; Rui Cerqueira; Adelino F. Leite-Moreira (1714-1719).
Angiotensin 1–7 is a bioactive heptapeptide of the renin–angiotensin system. Its cardiovascular actions have recently acquired growing relevance, mainly due to its counter-regulatory actions in the angiotensin cascade. The aim of the present study was to evaluate the actions of angiotensin 1–7 on myocardial function. Increasing concentrations of angiotensin 1–7 (10−9 to 10−5  M) were added to rabbit right papillary muscles: (1) in baseline conditions with intact endocardial endothelium (EE); (2) after selective removal of the EE with Triton X-100 (1 s, 0.01%); (3) with intact EE in the presence of the Mas receptor antagonist A-779, the AT1 receptor antagonist ZD-7155, the AT2 receptor antagonist PD-123,319 or the nitric oxide synthesis inhibitor NG-nitro-l-arginine (l-NA). Concerning the effects on contractility, we observed a significant decrease on active tension, dT/dt max, peak shortening and dL/dt max of −10.5 ± 3.6%, −8.0 ± 3.0%, −5.3 ± 2.6% and −5.7 ± 2.3%, respectively. There was no change on relaxation parameters, namely dT/dt min or dL/dt min. Time to half relaxation was significantly decreased. The presence of ZD-7155 or PD-123,319 did not change these effects. However, angiotensin 1–7 effects on myocardial properties were abolished after selective EE removal and in the presence of A-779 or l-NA. In conclusion, in this animal species, angiotensin 1–7 through its binding to Mas receptor induces a negative inotropic effect modulated by the EE and nitric oxide and independent of AT1 or AT2 receptors activation. As the effects described in the present work were influenced by the endocardial endothelium, they may be disrupted in situations associated to endothelial dysfunction, as in heart failure or myocardial ischemia.
Keywords: Angiotensin 1–7; Myocardial function; Contractility; Relaxation;

Regulation of ANP secretion from isolated atria by prostaglandins and cyclooxygenase-2 by Guanyi Bai; Shan Gao; Amin Shah; Kuichang Yuan; Woo Hyun Park; Suhn Hee Kim (1720-1728).
Cyclooxygenase (COX) is a key enzyme regulating the production of various prostaglandins (PGs) from arachidonic acid. Angiotensin II has been reported to be an important inflammatory mediator, which increases COX-2. The aim of this study was to determine the role of various PGs and COX-2 in the regulation of atrial natriuretic peptide (ANP) secretion. PGF2α and PGD2 caused dose-dependent increases in ANP release and intra-atrial pressure. The potency for the stimulation of ANP secretion by PGF2α was higher than that by PGD2. In contrast, PGE2, PGI2, PGJ2, and thromboxane A2 did not show any significant effects. The increases in intra-atrial pressure and ANP secretion induced by PGF2α and PGD2 were significantly attenuated by the pretreatment with an inhibitor of PGF2α receptor. By the pretreatment with an inhibitor for phospholipase C (PLC), inositol 3-phosphate (IP3) receptor, protein kinase C (PKC), or myosin light chain kinase (MLCK), PGF2α-mediated increase in ANP secretion and positive inotropy were attenuated. Inhibitor for COX-1 or COX-2 did not cause any significant effects on atrial parameters. In hypertrophied rat atria, PGF2α-induced positive inotropy and ANP secretion were markedly attenuated whereas COX-2 inhibitor stimulated ANP secretion. The expression of COX-2 increased and the expression of PGF2α receptor mRNA decreased in hypertrophied rat atria. These results suggest that PGF2α increased the ANP secretion and positive inotropy through PLC–IP3–PKC–MLCK pathway, and the modulation of ANP secretion by COX-2 inhibitor and PGF2α may partly relate to the development of renal hypertension.
Keywords: Cyclooxygenase; Prostaglandin; Hypertension; Signal transduction; ANP;

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is proteolytically activated by certain endogenous proteases, such as trypsin, tryptase, and factor Xa. PAR2 can also be activated by synthetic peptides if their sequence mimics the tethered ligand exposed after receptor cleavage. Although it is known that PAR2 modulates vascular reactivity, it is unclear whether at the chronic stage of type 2 diabetes there are alterations in PAR2-mediated vascular responses. We investigated this issue by exposing mesenteric artery rings to PAR2-activating peptide (PAR2-AP; SLIGRL-NH2), the arteries used being obtained from later-stage (32–40-week-old) type 2 diabetic Goto–Kakizaki (GK) rats. The PAR2-AP-induced relaxation was enhanced in GK rats (vs. age-matched Wistar rats), whereas the ACh-induced relaxation was weaker in GK than in Wistar rats. In both groups, the PAR2-AP-induced relaxation was largely blocked by endothelial denudation or by NG-nitro-l-arginine [nitric oxide (NO) synthase inhibitor] treatment, but it was unaffected by indomethacin (cyclooxygenase inhibitor) treatment. Both the NO production induced by PAR2-AP and the PAR2 protein expression were significantly increased in mesenteric arteries from GK rats (vs. Wistar rats). These data are the first to indicate that the PAR2-AP-induced endothelium-dependent relaxation is enhanced in mesenteric arteries isolated from type 2 diabetic GK rats at the chronic stage, and they further suggest that the enhancement may be due to an increased expression of PAR2 receptors in this artery.
Keywords: Endothelium-dependent relaxation; GK rat; Mesenteric artery; NO; PAR2; Type 2 diabetes;

Glucagon-like peptide-1 protects mesenteric endothelium from injury during inflammation by Kristopher C. Dozier; Elizabeth L. Cureton; Rita O. Kwan; Brian Curran; Javid Sadjadi; Gregory P. Victorino (1735-1741).
Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability (L p) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L p was measured. Second, after systemic LPS injection, L p was measured after subsequent perfusion with GLP-1. Thirdly, L p was measured after LPS injection and perfusion with GLP-1 + GLP-1 receptor antagonist. Lastly, L p was measured after LPS injection and perfusion with GLP-1 + inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC) ± SEM. GLP-1 had no effect on L p (AUC: baseline = 27 ± 1.4, GLP-1 = 1 ± 0.4, p  = 0.08). LPS increased L p two-fold (AUC: LPS = 54 ± 1.7, p  < 0.0001). GLP-1 reduced the LPS increase in L p by 75% (AUC: LPS + GLP-1 = 34 ± 1.5, p  < 0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + antagonist = 46 ± 2.0, p  < 0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + cAMP inhibitor = 46 ± 1.5, p  < 0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS + GLP-1 + PKA inhibitor = 56 ± 1.5, p  < 0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.
Keywords: Microvascular permeability; Glucagon-like peptide-1; GLP-1;

Association of obestatin with blood pressure in the third trimesters of pregnancy by An-Jing Ren; Qian He; Jing-Song Shi; Zhi-Fu Guo; Xing Zheng; Li Lin; Yang-Kai Wang; Song-Yun Xia; Li-Li Sun; Xin Du; Ying Sun; Lan-Mei Zhang; Wen-Jun Yuan (1742-1745).
Obestatin is a recently discovered 23-amino acid peptide encoded by the same gene that encodes ghrelin. It has been reported that there is a significant negative correlation between the plasma ghrelin concentration and systemic blood pressure in patients with pregnancy-induced hypertension. We investigated the plasma concentration of obestatin in 18 non-pregnant women, 18 normal pregnant women, and 15 patients with pregnancy-induced hypertension. The plasma concentrations of obestatin in these 3 groups of women were 63.4 ± 9.5 pg/ml, 38.1 ± 6.3 pg/ml, and 46.0 ± 9.3 pg/ml, respectively. In non-pregnant women, there was no correlation between the plasma obestatin concentration and the mean arterial pressure. However, there was a positive correlation between the plasma obestatin concentration and the mean arterial pressure in normal pregnant women and pregnant women with pregnancy-induced hypertension. These results suggest that obestatin may have some potential role in the regulation of blood pressure in normal pregnant women and women with pregnancy-induced hypertension.
Keywords: Obestatin; Ghrelin; Pregnancy; Hypertension;

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells by Yi-Min Zhang; Jun Peng; Chang-Ping Hu; Qiu-Tao Jiang; Guo-Long Jiang; Yuan-Jian Li (1746-1752).
The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α2-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α2-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α2-adrenoceptor, which is related to the NO pathway.
Keywords: α2-Adrenoceptor; Calcitonin gene-related peptide; Clonidine; Nitric oxide;

This review focuses on the evolutionary and functional relationship of calcitonin receptor-stimulating peptide (CRSP) with calcitonin (CT)/calcitonin gene-related peptide (CGRP) in mammals. CRSP shows high sequence identity with CGRP, but distinct biological properties. CRSP genes (CRSPs) have been identified in mammals such as pigs and dogs of the Laurasiatheria, but not in primates and rodents of the Euarchontoglires or in non-placental mammals. CRSPs have genomic organizations highly similar to those of CT/CGRP genes (CT/CGRPs), which are located along with CGRPs in a locus between CYP2R1 and INSC, while the other members of the CGRP superfamily, adrenomedullin and amylin, show genomic organizations and locations distinct from CT, CGRP, and CRSP. Thus, we categorized these three peptides into the CT/CGRP/CRSP family. Non-placental mammals having one and placental mammals having multiple CT/CGRP/CRSP family genes suggests that multiplicity of CT/CGRP started at an early stage of mammalian evolution. In the placental mammals, Laurasiatheria generally possesses multiple CRSPs and only one CT/CGRP, while Euarchontoglires possesses CT/CGRP and CGRPβ but no CRSP, indicating an increase in the diversity and multiplicity of this family of genes in mammalian evolution. Phylogenetic analysis suggests that some CRSPs have been generated very recently in mammalian evolution. Taken together, the increase in the number and complexity of the CT/CGRP/CRSP family genes may have due to evolutionary pressure to facilitate adaptation during mammalian evolution. In this regard, it is important to elucidate the physiological roles of CT, CGRP and CRSP from the viewpoint of the CT/CGRP/CRSP family even in Euarchontoglires.
Keywords: Calcitonin receptor-stimulating peptide; Calcitonin gene-related peptide; Calcitonin; Phylogenetic analysis; Evolution;

Regulatory mechanism of the arginine vasopressin-enhanced green fluorescent protein fusion gene expression in acute and chronic stress by Hitoshi Suzuki; Makoto Kawasaki; Hideo Ohnishi; Toshitaka Nakamura; Yoichi Ueta (1763-1770).
Various kinds of stress cause neuroendocrine responses such as corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) release from parvocellular division of the paraventricular nucleus (PVN) and activation of the hypothalamo-pituitary adrenal (HPA) axis. We examined the effects of acute and chronic stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using chronic salt loading as an osmotic stimulation, intraperitoneal administration of lipopolysaccharide (LPS) as acute inflammatory stress and adjuvant arthritis (AA) as chronic inflammatory/nociceptive stress. Salt loading caused a marked increase in the eGFP gene expression and eGFP fluorescence in the supraoptic nucleus, magnocellular division of the PVN and internal layer of the median eminence (ME). Administration of LPS caused increased fluorescence in parvocellular division of the PVN and external layer of the ME. AA rats revealed an increased expression of the eGFP gene and eGFP fluorescence in both magnocellular and parvocellular divisions of the PVN and both internal and external layers of the ME. On the other hand, the levels of the CRH gene expression in parvocellular division of the PVN were significantly decreased as AA developed, though plasma concentrations of corticosterone were significantly increased. These results indicate that AVP-eGFP transgenic rats enable the detection of changes in AVP expression more easily than by using procedures such as immunohistochemistry. We propose that AVP-eGFP transgenic rats represent a useful animal model for further understanding of the physiology of AVP expression in the hypothalamo-pituitary system under various physiological conditions, including various kinds of stress.
Keywords: Stress; Hypothalamo-pituitary adrenal axis; Arginine vasopressin; Corticotropin-releasing hormone; GFP;

The effect of exenatide re-exposure on safety and efficacy by Peter Faludi; Robert Brodows; Jude Burger; Tibor Ivanyi; Daniel K. Braun (1771-1774).
Exenatide, a synthetic peptide originally isolated from salivary secretions of Heloderma suspectum, like other subcutaneously injected peptides, can cause antibody formation. Despite that antibody formation has been observed in some patients, results from previous clinical trials have not shown safety and efficacy concerns in exenatide-naïve patients. The objective of this multicenter, open-label study was to investigate the response of anti-exenatide antibody formation and the incidence of immune-related and hypersensitivity reactions after exenatide re-exposure. Fifty-eight patients (57% male; 59 ± 10 years; weight 85 ± 19 kg; HbA1c 8.1 ± 0.9%; duration of diabetes 10 ± 5 years) were enrolled. At study initiation, 98.3% of patients were taking 1 or more antidiabetes drugs, including oral medication and various types of insulin. Treatment-emergent adverse events (TEAEs) at any time during the study were observed in 40 and 47% of patients with positive and negative treatment-emergent antibodies, respectively. Immune-related AEs were observed in 6 patients (4 were antibody positive). These AEs had not been reported in their previous exposure to exenatide. Re-exposure to exenatide did not result in increased hypersensitivity reactions. Overall, 72% of patients had a baseline to endpoint reduction in HbA1c (range −0.1 to −2.8%), and 87% of antibody negative versus 62% of antibody positive patients had an HbA1c endpoint reduction. The study design and the patients’ baseline characteristics, including diabetes treatment at study initiation, are confounding factors limiting clinical conclusions on exenatide's glycemic effect in this patient population. The study results indicate that anti-exenatide antibody formation did not increase the incidence of TEAEs in patients re-exposed to exenatide.