Peptides (v.26, #3)

Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction by Scott F. Cummins; Amy E. Nichols; Carrie J. Warso; Gregg T. Nagle (351-359).
Mate attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, and temptin which are released during egg-laying. Other water-borne pheromones are predicted to act in concert with attractin, enticin, and temptin, but their identities were unknown. We recently identified a highly expressed Aplysia californica albumen gland gene (Alb-23) that encoded a novel protein by differential library screening of an albumen gland cDNA library. To determine whether Alb-23 (‘seductin’) was a water-borne pheromone, we employed Western blot analysis, purification and expression of albumen gland proteins, immunolocalization studies, pheromone secretion assays, comparative genomics, and behavioral bioassays. Immunoreactive seductin was detected in eluates of egg cordons, indicating that seductin was secreted onto the cordon during egg laying. Aplysia brasiliana seductin was 94% identical to its A. californica homolog. In T-maze attraction assays, the combination of attractin and seductin was significantly attractive to potential mates, whereas either protein alone was not. Data from this and previous studies support the hypothesis that seductin is a water-borne protein pheromone that acts in concert with attractin, enticin, and temptin to attract Aplysia to form and maintain mating aggregations.
Keywords: Aplysia; Protein pheromone; Seductin; Attractin; Enticin; Temptin;

Direct cDNA cloning of novel conopeptide precursors of the O-superfamily by Silke Kauferstein; Christian Melaun; Dietrich Mebs (361-367).
Conotoxins from the venom of marine cone snails (genus Conus) represent large families of proteins exhibiting a similar precursor organization, but highly diverse pharmacological activities. A directed PCR-based approach using primers according to the conserved signal sequence was applied to investigate the diversity of conotoxins from the O-superfamily. Using 3′ RACE, cDNA sequences encoding precursor peptides were identified in five Conus species (Conus capitaneus, Conus imperialis, Conus striatus, Conus vexillum and Conus virgo). In all cases, the sequence of the signal region exhibited high conservancy, whereas the sequence of the mature peptides was either almost identical or highly divergent among the five species. These findings demonstrate that beside a common genetic pattern divergent evolution of toxins occurred in a highly mutating peptide family.
Keywords: Conotoxin; O-superfamily; cDNA cloning; Evolution; Conopeptides; Cone snails;

Deletion of two C-terminal Gln residues of 12–26-residue fragment of melittin improves its antimicrobial activity by Xuejun Sun; Suxia Chen; Shunzi Li; Husheng Yan; Yunge Fan; Huaifeng Mi (369-375).
In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12–26) (GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction with bacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12–25) and Mel(12–24), were synthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12–26). If both of the Gln residues of Mel(12–26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increased slightly. If the Gln residue of Mel(12–25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, although the substituted peptide possessed much higher hydrophobicity and higher α-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminal residues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletion may be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes because of the lower molecular weights of the deletion peptides.
Keywords: Melittin; Antimicrobial peptide; Antibiotic; Hemolysis;

Prokineticins are small (∼8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (M r  = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.
Keywords: Amphibian; Venom; Mass spectrometry; Protein; Cloning;

Cholecystokinin mRNA in Atlantic herring, Clupea harengus—molecular cloning, characterization, and distribution in the digestive tract during the early life stages by Yuko Kamisaka; Øyvind Drivenes; Tadahide Kurokawa; Masatomo Tagawa; Ivar Rønnestad; Masaru Tanaka; Jon Vidar Helvik (385-393).
The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.
Keywords: Cholecystokinin; Cloning; Distribution; Digestive tract; Herring; In situ hybridization; Juvenile; Larvae; Ontogeny;

Glyoxalase I activity has been shown to be directly related to cancer and its inhibitors have been used as anti-cancer drugs. Immunochemical studies have shown immunochemical relatedness among animal and plant glyoxalase I, but its potential application for biomedical research has not been investigated. In order to understand the conserved immunochemical regions of the protein and to determine probable immunomodulation targets, a cellulose-bound scanning peptide library for Brassica juncea glyoxalase I was made using the spot synthesis method. Immuno-probing of the library, using B. juncea anti-glyoxalase I monospecific polyclonal antibodies, revealed three immunodominant regions, epitope I, II, and III. In the homology model of B. juncea glyoxalase I generated by threading its sequence onto the human glyoxalase I, the high accessible surface area and the hydrophilic nature of the epitopes confirmed their surface localization and hence their accessibility for antigen–antibody interaction. Epitopes I and II were specific to B. juncea glyoxalase I. Localizing the epitopes on available glyoxalase I sequences showed that epitope III containing the active site region was conserved across phyla. Therefore, this could be used as a potential immunomodulation target for cancer therapy. Moreover, as the most immunogenic epitopes were mapped on the surface of the protein, this method could be used to discover potential therapeutic targets. It is a simple and fast approach for such investigations. This study, to our knowledge, is the first in epitope mapping of glyoxalase I and has great biomedical potential.
Keywords: Brassica juncea glyoxalase I; Epitope-mapping; Epitope localization; Immunomodulation; Peptide library; Molecular modeling;

The inhibitory effects of synthetic short peptides, mimicking MICA and targeting at NKG2D receptors, on function of NK cells by Bin Zhang; Haiming Wei; Xiaodong Zheng; Jian Zhang; Rui Sun; Zhigang Tian (405-412).
NKG2D is an activating receptor expressed on most of human NK cells, one of whose ligands is MICA. Based on the crystal structure of NKG2D–MICA complex, we synthesized three short peptides (P1, P2 and P3), mimicking functional α1 and α2 domain of MICA. The inhibitory effects of three peptides on NK-92 cells, a human NK cell line against Hela cells were observed and the inhibitory percentage was 38% at maximum for P1 + P2 + P3 in concentration of 1 nM. The same peptides had no effect on NK-92 cell against target cells lacking MICA (K562 cells line). The unrelated peptides as controls had no effect on the system. Two peptides (P2 and P3) were prolonged at one or both ends, and the longer forms of peptides exerted stronger inhibitory effects than their shorter forms. Each combination of two peptides exerted a stronger function than single peptide (P1, P2, P3), indicating that shedding of longer amino acid sequence of α1 domain or more domain sites of MICA are better than shorter sequence and fewer sites. P1 + P2 + P3 revealed the almost same inhibitory rate as the soluble MICA (sMICA). P1 + P2 + P3 were also able to alleviate the concanavalin A-induced murine autoimmune hepatitis in vivo, conforming the similarity of NKG2D between human and mice. The results demonstrate that MICA-mimicking peptides will be useful to search the specific functional sites for NKG2D–MICA interaction, but also promising in explaining NKG2D-related autoimmunity.
Keywords: NK cells; Natural cytotoxicity; NKG2D; MICA; Peptide; Autoimmune liver injury;

Radioprotection by N-palmitoylated nonapeptide of human interleukin-1β by Vijay K. Singh; Venkataraman Srinivasan; Thomas M. Seed; William E. Jackson; Venita E. Miner; K. Sree Kumar (413-418).
Interleukin-1β (IL-1β) is a cytokine involved in homeostatic processes of the immune system and specifically in inflammatory reactions. The nonapeptide of human IL-1β (VQGEESNDK, position 163–171) has been shown to retain adjuvant and immunostimulatory activities of the native molecule without any inflammatory and pyrogenic properties. A lipophilic derivative of IL-1β nonapeptide having a palmitoyl residue at the amino terminus was synthesized in order to determine the effects of such structural modification on its bioactivities. The structurally modified peptide derivative, palmitoylated peptide, significantly protected C3H/HeN mice against potentially lethal doses of ionizing radiation. The dose reduction factor was found to be 1.07. Hematological studies show improved recovery of red blood cells and platelets in irradiated and palmitoylated peptide treated mice as compared with the untreated and irradiated group. These results suggest the importance of the derivatization of small peptides of radioprotective, but toxic cytokines in order to enhance radioprotective activity while reducing unwanted toxic side effects.
Keywords: Radioprotection; Hematology; Palmitoylated peptide;

Importance of the central region of lamprey gonadotropin-releasing hormone III in the inhibition of breast cancer cell growth by Krisztina Herédi-Szabó; Jeremiah Lubke; Geza Toth; Richard F. Murphy; Sándor Lovas (419-422).
Naturally occurring isoforms of the decapeptide gonadotropin-releasing hormone (GnRH) share residues 1–4 and 9–10. lGnRH-III, the third isoform isolated in the sea lamprey has no endocrine effect in mammals but shows a direct antiproliferative effect on human breast, prostate and endometrial cancer cell lines. To investigate these features, residues 5–8 of lGnRH-III were systematically replaced with Ala. The ability of the synthetic analogs to interact with receptors on MDA-MB 231 human breast cancer cells and their effect on the growth of the same cell line were investigated.[Ala6]lGnRH-III and [Ala7]lGnRH-III have neither receptor binding nor antiproliferative activity. Replacement of His5 with Ala resulted in an analog that binds to the receptor but does not have antiproliferative activity. The results are in agreement with previous reports that modifications of Lys at position 8 are well tolerated.
Keywords: lGnRH-III; Ala-scan; Receptor binding; Cancer growth; Specific growth inhibition; MDA-MB 231;

Pharmacodynamics and pharmacokinetics of recombinant hirudin via four non-parenteral routes by Yu Liu; Wan-liang Lu; Xuan Zhang; Xue-qing Wang; Hua Zhang; Qiang Zhang (423-430).
One of recombinant hirudin variants, rHV2, a polypeptide used as an anticoagulant agent in clinic, was administered to anesthetized rats via intratracheal, buccal, nasal and rectal routes. Prolongation in clotting time and thrombin time was measured to calculate pharmacological bioavailability. Plasma concentration of rHV2 was determined using a chromogenic thrombin substrate assay and pharmacokinetic parameters were obtained on the basis of a non-compartmental model. Intravenous administration was also performed as the gold standard by which the other routes were compared. Difference in pharmacological bioavailability (P.A.), bioavailability (F) and absorption rate of rHV2 was found for the four non-parenteral routes. The rank order for both P.A. and F was intratracheal > nasal > buccal > rectal. Absorption was more rapid after both intratracheal and rectal administration (t max  ∼ 20–40 min), compared with that after nasal and rectal administration. It is evident that the pulmonary route is preferable to other three routes for successful systemic delivery of rHV2.
Keywords: Absorption; Recombinant hirudin; Non-parenteral route; Pharmacodynamics; Pharmacokinetics;

Antinociceptive action of hemopressin in experimental hyperalgesia by Camila Squarzoni Dale; Rosana de Lima Pagano; Vanessa Rioli; Stephen Hyslop; Renata Giorgi; Emer Suavinho Ferro (431-436).
Endogenous hemorphins, derived from degradation of the β-chain of hemoglobin, lower arterial blood pressure and exert an antinociceptive action in experimental models of nociception. Hemopressin, derived from the α-chain of hemoglobin, also decreases blood pressure, but its effects on pain have not been studied. In this work, we examined the influence of hemopressin on inflammatory pain. Hemopressin reverted the hyperalgesia induced by either carrageenin or bradykinin when injected concomitantly or 2.5 h after the phlogistic agents. Hemopressin administered systemically also reverted the hyperalgesia induced by carrageenin. Naloxone did not prevent the antinociceptive action of this peptide. These data suggest that hemopressin inhibits peripheral hyperalgesic responses by mechanisms independent of opioid receptor activation.
Keywords: Antinociception; Carrageenin; Hemopressin; Hyperalgesia; Rats;

Effect of CCK-8 on insulin-induced hyperphagia and hypothalamic orexigenic neuropeptide expression in the rat by Eva Gallmann; Denis Arsenijevic; Marianne Spengler; Gareth Williams; Wolfgang Langhans (437-445).
The influence of cholecystokinin octapeptide (CCK-8) on normal and insulin-induced feeding and expression of orexigenic hypothalamic neuropeptides was investigated in male rats. CCK-8, administered during meals (4 μg/kg) or continuously (32 μg/kg over 60 min), blunted the stimulating effect of insulin (50 IU/kg) on feeding by reducing meal size (−60%; P  < 0.05 or −86%; P  < 0.0001, respectively). Rats without access to food and injected with IP insulin (50 IU/kg) showed increased hypothalamic mRNA levels of orexin (+30%; P  < 0.05) and melanin-concentrating hormone (+52%; P  < 0.05), as compared with ad libitum-fed and saline-injected control rats. Continuous IP infusion of CCK-8 (32 μg/kg) blunted these increases. Our results suggest that both orexin and melanin-concentrating hormone participate in the response to insulin hypoglycemia without food being present; these neurons may be involved in mechanisms related to insulin-induced hyperphagia. Signals triggered by peripheral CCK-8 act to decrease the expression of orexin and melanin-concentrating hormone. This may be associated with a reduction in hyperphagia.
Keywords: Food intake; Satiety signals; Brain; Hypoglycemia;

The effect of lipopolysaccharide on cholecystokinin in murine plasma and tissue by Tracey J. Weiland; Stephen Kent; Nicholas J. Voudouris; Arthur Shulkes (447-455).
Several mechanisms have been proposed for neuroimmune communication supporting sickness behavior (fever, anorexia, inactivity, and cachexia) following infection. We examined the role of cholecystokinin as a neurochemical intermediary of sickness behavior by determining plasma, duodenum, hypothalamus, and brainstem cholecystokinin concentrations 30 and 60 min and 12 h following intraperitoneal lipopolysaccharide (LPS) (0.25 and 2.5 mg/kg). Hypothalamic cholecystokinin was significantly lower in LPS- versus saline-treated mice 30 min (0.25 and 2.5 mg/kg) and 12 h (2.5 mg/kg) post-injection. Plasma cholecystokinin of LPS-treated mice was significantly lower than that of controls 1 and 12 h post-injection, a finding consistent with a non-endocrine action of peripheral cholecystokinin.
Keywords: Cholecystokinin; Duodenum; Hypothalamus; Lipopolysaccharide; Sickness behavior; Cytokines;

The CRF2 receptor is involved in stress responses, cardiovascular function and gastric motility. Endogenous agonists (urocortin (UCN) 2, UCN 3) and synthetic antagonists (astressin2-B, antisauvagine-30) are selective for CRF2 over the CRF1 receptor. Peptide ligand binding properties of the CRF2 receptor require further investigation, including ligand affinity for endogenously expressed receptors, the effect of receptor–G-protein coupling on ligand affinity, and the molecular basis of ligand selectivity. Ligand affinity for rat CRF2(a) in olfactory bulb and CRF2(b) in A7r5 cells was similar to that for the cloned human CRF2(a) receptor (within three-fold), except for oCRF (9.4- and 5.4-fold higher affinity in olfactory bulb and A7r5 cells, respectively). Receptor–G-protein uncoupling reduced agonist affinity only 1.2- to 6.5-fold (compared with 92–1300-fold for the CRF1 receptor). Ligand selectivity mechanisms were investigated using chimeric CRF2/CRF1 receptors. The juxtamembrane receptor domain determined selectivity of antisauvagine-30, the N-terminal-extracellular domain contributed to selectivity of UCN 3, and both domains contributed to selectivity of UCN 2 and astressin2-B. Therefore ligands differ in the contribution of receptor domains to their selectivity, and CRF2-selective antagonists bind the juxtamembrane domain. These findings will be important for identifying the CRF2 receptor in tissues and for developing ligands targeting the receptor, both of which will be useful in identifying the emerging physiological functions of the CRF2 receptor.
Keywords: Corticotropin-releasing factor; G-protein-coupled receptor; Urocortin; Binding; Sauvagine; Astressin;

Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro by Masaru Ishibashi; Shinobu Takano; Hiroki Yanagida; Masafumi Takatsuna; Kazuki Nakajima; Yutaka Oomura; Matthew J. Wayner; Kazuo Sasaki (471-481).
Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX1R) and orexin 2 (OX2R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca2+ concentration ([Ca2+] i ) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10−8 to 10−6  M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10−7  M. When both ORX-A and ORX-B were applied simultaneously at 10−7  M, the increase in firing rate was almost equal to that of ORX-B at 10−7  M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX1R receptors. The excitatory effect of ORX-B was seen in low Ca2+ and high Mg2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca2+ and high Mg2+ ACSF than in normal ACSF. [Ca2+] i imaging studies demonstrated that [Ca2+] i was increased in about 50% of the PVT neurons by both 10−7  M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca2+] i induced by ORX-B was abolished in Ca2+-free ACSF, suggesting that ORX-B does not release Ca2+ from intracellular Ca2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca2+-free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca2+-free and TTX-containing Ca2+-free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about −97 mV, and an increase of extracellular K+ concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K+ channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX2R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors.
Keywords: Paraventricular nucleus of the thalamus; PVT; Orexin-A/hypocretin-1; Orexin-B/hypocretin-2; Calcium; Potassium channel; Patch clamp; [Ca2+] i imaging;

Oxytocin ameliorates oxidative colonic inflammation by a neutrophil-dependent mechanism by Sevgin Özlem İşeri; Göksel Şener; Beyhan Sağlam; Nursal Gedik; Feriha Ercan; Berrak Ç. Yeğen (483-491).
Oxytocin (OT), a nonapeptide produced in the paraventricular and the supraoptical nuclei in the hypothalamus has a wide range of effects in the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. OT may participate in the regulation of motility, secretion, blood flow, cell turnover and release of neurotransmitters and/or peptides in the GI tract, possesses antisecretory and antiulcer effects, facilitates wound healing and is involved in the modulation of immune and inflammatory processes. The present work was conducted to assess the possible therapeutic effects of OT against the acetic acid-induced colonic injury in the rat. Methods: Colitis was induced by intracolonic administration of acetic acid (5%) in Sprague–Dawley rats (200–250 g). Either saline or OT (0.5 mg/kg) was injected subcutaneously, immediately after the induction of colitis and repeated two times a day for 4 days. On the 4th day, rats were decapitated and distal 8 cm of the colon were removed for the macroscopic and microscopic damage scoring, determination of tissue wet weight index (WI), malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. Colonic collagen content, as a fibrosis marker was also determined. Lactate deydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) levels were assayed in serum samples. In the acetic acid-induced colitis, macroscopic and microscopic damage scores, WI, MDA and MPO levels were significantly increased, while GSH levels were decreased when compared to control group (p  < 0.05–<0.001). Treatment with OT abolished the colitis-induced elevations in damage scores, WI, MDA and MPO levels and restored the GSH levels (p  < 0.05–0.001). Similarly, acetic acid increased the collagen content of colonic tissues and OT-treatment reduced this value to the level of the control group. Serum LDH and TNF-α levels were also elevated in the acetic acid-induced colitis group as compared to control group, while this increase was significantly decreased by OT treatment. The results suggest that OT, which improves the antioxidative state of the colonic tissue and ameliorates oxidative colonic injury via a neutrophil-dependent mechanism, requires further investigation as a potential therapeutic agent in colonic inflammation.
Keywords: Oxytocin; Colitis; Oxidative damage; Inflammation; Neutrophils;

Octreotide ameliorates sepsis-induced pelvic inflammation in female rats by a neutrophil-dependent mechanism by Göksel Şener; Şule Çetinel; Gözde Erkanlı; Nursal Gedik; Berrak Ç. Yeğen (493-499).
Sepsis is a generalized inflammatory response, which involves organ systems remote from the locus of the initial infectious insult, accompanied by the release of cytokines and the subsequent formation of reactive oxygen and nitrogen species. The aim of this study was to investigate the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against sepsis-induced oxidative damage in the uterine and ovarian tissues of rats. Sepsis was induced by caecal ligation and puncture method in female Wistar albino rats. Sepsis and sham operated (control) groups received either saline or OCT (50 μg/kg, i.p.; Novartis) immediately after the operation and at 12 h. Twenty-four hours after the surgery, rats were decapitated and serum TNF-α levels and tissue malondialdehyde (MDA) content, glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the uterus and ovaries. Oxidant-induced tissue fibrosis was determined by tissue collagen contents, while the extent of tissue injuries was analyzed microscopically. Sepsis increased serum TNF-α levels and resulted in decreased GSH levels and increased MDA levels, MPO activity and collagen contents in both the uterus and the ovaries (p  < 0.05–0.001) indicating the presence of the oxidative damage, as also confirmed by histological analysis. On the other hand, OCT administration reversed these oxidant responses and reduced the severity of microscopic damage (p  < 0.001). In conclusion, OCT protects against sepsis-induced oxidative injury of the uterine and ovarian tissues by diminishing neutrophil infiltration, an important source of oxygen free radicals. Our results suggest that OCT may be of therapeutic value in ameliorating sepsis-associated pelvic inflammation.
Keywords: Sepsis; Octreotide; Neutrophil; Uterine; Ovary;

Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts by Jing-Hui Yang; Yong-Fen Qi; Yue-Xia Jia; Chun-Shui Pan; Jing Zhao; Jun Yang; Jaw-Kang Chang; Chao-Shu Tang (501-507).
Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate a 47-amino acid mature peptide (IMD1–47) and a shorter 40-amino acid one (IMD8–47) by proteolytic cleavage. The present study was designed to determine the protective effect of IMD on cardiac ischemia/reperfusion (I/R) injury and its possible mechanism. Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 45-min global ischemia and 30-min reperfusion. Cardiac function was measured. The release of myocardial protein and lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) were assayed. Myocardial cAMP content was determined by radioimmunoassay (RIA). Cardiac I/R induced a marked inhibition of cardiac function and myocardial injury. Reperfusion with IMD significantly attenuated the I/R injury. Compared with I/R alone, perfusion with 10−8  mol/L IMD1–47 and IMD8–47 induced a 36% and 33% increase in Δ left ventricular pressure (ΔLVP), 30% and 28% in maximal rate of increase of LV pressure (+LVdP/dt max), and 34% and 31% in maximal rate of decrease of LV pressure (−LVdP/dt max), respectively (all P  < 0.01) but an approximately 58% and 51% decrease in LV diastolic pressure, respectively (P  < 0.01). In addition, perfusion with IMD markedly attenuated the leakage of LDH, total protein and myoglobin from myocardia compared with I/R alone. The contents of ventricular myocardia cAMP after reperfusion with 10−8  mol/L IMD1–47 and IMD8–47 were 130% and 91% higher, respectively, than that with I/R alone (all P  < 0.01). However, formations of myocardial MDA were 52% and 50% lower than that with I/R alone (all P  < 0.01), respectively. Interestingly, the above IMD effects were similar to those of adrenomedullin (10−8  mol/L). These results suggest that IMD, like adrenomedullin, exerts cardio-protective effects against myocardial I/R injury.
Keywords: Intermedin; Ischemia/reperfusion; cAMP; Heart;

All d-VIP mitigates vasodilation elicited by l-VIP, micellar l-VIP and micellar PACAP1–38, but not PACAP1–38, in vivo by Israel Rubinstein; Beena Ashok; Takaya Tsueshita; Hayat Önyüksel (509-515).
The purpose of this study was to determine whether all d-vasoactive intestinal peptide (VIP), an inactive optical isomer of l-VIP, modulates the vasorelaxant effects of human l-VIP and pituitary adenylate cyclase activating peptide (PACAP)1–38, two ubiquitous and pleiotropic neuropeptides that activate VPAC1 and VPAC2, two VIP subtype receptors, in the intact peripheral microcirculation. Using intravital microscopy, we found that suffusion of all d-VIP had no significant effects on arteriolar diameter in the intact hamster cheek pouch. However, all d-VIP significantly attenuated l-VIP-induced vasodilation in a concentration-dependent fashion (P  < 0.05). Likewise, all d-VIP significantly attenuated the vasorelaxant effects of l-VIP associated with sterically stabilized phospholipid micelles (SSM; P  < 0.05). Although all d-VIP had no significant effects on l-PACAP1–38-induced vasodilation, it abrogated PACAP1–38 in SSM-induced responses (P  < 0.05). The effects of all d-VIP were specific because it had no significant effects on acetylcholine-, nitroglycerin- and bradykinin-induced vasodilation. Taken together, these data indicate that all d-VIP attenuates the vasorelaxant effects of random coil and α-helix l-VIP as well as those of α-helix but not random coil PACAP in the intact peripheral microcirculation in a specific fashion. These effects are mediated, most likely, through interactions with VPAC1/VPAC2 receptors. We suggest that all d-VIP could be exploited as a novel, safe and active targeting moiety of VPAC1/VPAC2 receptors in vivo.
Keywords: Microcirculation; Neuropeptide; Optical isomer; VPAC1/VPAC2 receptors; Sterically stabilized phospholipid micelles; Targeting; Hamster;

Development of a selective peptide antagonist for the human natriuretic peptide receptor-B by Julie Deschênes; Cécile Duperé; Normand McNicoll; Nicholas L’Heureux; François Auger; Alain Fournier; André De Léan (517-524).
Activation by C-type natriuretic peptide (CNP) of its receptor NPRB results in venodilation and inhibition of cellular proliferation. NPRB-selective antagonists should be useful to understand their physiological implications. We previously observed that [Thr9,Ser11,Arg16](N,C-ANP)pBNP (P12) is an antagonist for bNPRB and a potent agonist for bNPRA. The antagonist [Ser11](N-CNP,C-ANP)pBNP(2-26) (P18) displays six-fold selectivity towards hNPRB versus hNPRA. Deletion of the C-terminus in [Ser11](N-CNP,C-ANP)pBNP(2-25) (P19) decreases its affinity for hNPRA but improves its selectivity 35-fold. Peptide libraries based on P19 using phage display methodology yielded two positive clones P20 and P21. P19 behaves as the most potent antagonist, but P20 is the most selective.
Keywords: Natriuretic peptides; Guanylyl cyclase-coupled receptors; Phage display; Blood pressure;

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.
Keywords: Fluorescence; Imaging; Calcium; Myocytes; Calcitonin gene related peptide;

Effect of corticosterone on CART peptide levels in rat blood by A. Vicentic; R.G. Hunter; M.J. Kuhar (531-533).
We have recently demonstrated that CART peptides display a diurnal rhythm in blood that depends partly on glucocorticoids levels. This study extends previous findings by directly testing the effects of acute administration of corticosterone and metyrapone on CART peptide levels in blood. Acute treatment with corticosterone augmented CART levels, while metyrapone administration prevented the increase in CART in the evening hours. These results further support the hypothesis that glucocorticoids play a role in the regulation of CART levels in blood.
Keywords: Corticosteroids; Metyrapone; Cocaine and amphetamine-regulated transcripts;