Figure 2. 5-HT2CR and c-Fos IR-positive cell number in the paraventricular nucleus (PVN)detected by immunohistochemical methods.

Su1911

Neuropeptide S: Effects on Motility, Contractility and Inflammation in the Ratand Human Gastrointestinal TractPer M. Hellström, Md Abdul Halim, Markus Sjöblom, Salman Saudi, Anna Sommansson,Evelina Rosenqvist, Linda Gillberg, Tobias Feldreich, Magnus Sundbom, Urban Karlbom,Erik Naslund, Dominic-Luc Webb

Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrinecells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked toincreased risk of inflammatory bowel disease as well as motor and sensory disturbances ofthe gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects onmotility and inflammation is needed. Methods: Studies of motility were carried out in ratswith electrodes implanted in the small bowel. NPS was infused IV for 60 min and effectson myoelectrical activity were recorded. Motility effects of NPS were further studied asluminal pressure changes in anaesthetized rats where the proximal small intestine with intactblood supply was perfused with saline for motility recordings. Tissue samples were obtainedfrom rats for evaluation of gene expression and protein elaboration of inflammatory biomark-ers. Muscle strips of the human stomach, small intestine and colon were used for pharmaco-logical analysis in organ baths of motility responses to NPS. Localization of the NPS receptorwas done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS weremeasured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: Inconscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1reduced spiking and increased the MMC cycle length (P=0.005). In anesthetized rats, NPS0.01-1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P<0.001). In rats,NPS also increased the mRNA expression of iNOS, and CXCL1 and IL-1β at the proteinlevel. In human, fluorescent protein-tagging showed the NPS receptor primarily to be presentin enolase-positive nerve fibers, but some scattered also in muscle tissue. In organ baths,NPS 1-100 nM caused TTX-dependent relaxations of the smooth muscle. We found nodetectable levels of NPS in plasma of neither healthy subjects nor IBD patients. Conclusion:NPS has a smooth muscle-relaxing effect in the GI tract and dampens myoelectrical andcontractile motor activity of the small bowel. This is achieved primarily through an inhibitionof nerve-mediated relaxation of smooth muscle cells. Circulating NPS levels in humans areundetectable in healthy subjects and in active IBD. This implies that NPS exerts its inhibitoryfunction by a paracrine or neurocrine mechanism in the gut. NPS seems also capable ofintervening with inflammation by increasing gut cytokines and iNOS expression.

Su1912

μ Opioid Receptor Activation Protects Against Intestinal Ischemia-ReperfusionInjury in Mice Through Different Pathways During Early and Late Phase ofReperfusionCeline C. Duraffourd, Jessica Tsui, Nick Brecha, Catia Sternini

Background: Intestinal ischemia is a life-threatening emergency that can occur with abdominalsurgery and bowel transplantation. Tissue injury caused by ischemia and reperfusion (I/R)involves oxidative stress, activation of mitogen activated protein kinase (MAPK), and induc-tion of inflammatory genes initiating local inflammation. We have shown that activation ofperipheral μ opioid receptors (μORs) ameliorates tissue injury induced by I/R in the early-reperfusion stage by reducing neutrophil infiltration and cytokine expression. Aims: To testthe effect of μOR activation in early vs. late phase of I/R, and which pathways are involvedin μOR protection. Methods: I/R was induced in C57BL/6 mice by occlusion of the superiormesenteric artery (45 min) followed by 5 hr (I/R5) or 24 hr (I/R24) reperfusion. I/R, shamoperated (SO) and control (CTL) mice were treated with saline or the μOR agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO, 0.02 mg/kg) with or without the μOR antagonist[H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] (CTAP; 0.2 mg/kg) s.c. 20 min beforeischemia, and at 2 hr after the beginning of reperfusion. Intestinal inflammation was evaluatedusing a myeloperoxidase activity assay (MPO) and mucosal damage. TNFα mRNA a cytokinethat plays a prominent role in I/R inflammation, was measured by qRT-PCR. The expressionof proteins modulating inflammatory responses including nuclear factor kappa-B (NF-kB),stress responsive protein Heme Oxygenase-1 (HO-1), antiapoptotic factor, Bcl-xl, and MAPK/ERK (extracellular regulated kinase1/2) signaling pathway, were evaluated using Westernblot. Results: I/R mice developed mucosal damage, neutrophil infiltration, and increased

S-499 AGA Abstracts

TNFα mRNA compared to SO and CTL mice, with higher levels at I/R 5h than I/R 24h. NF-kB was also increased in I/R5, but most prominently in I/R24. DAMGO reduced inflammatoryindexes and upregulated HO-1 and ERK1/2 in the early period of reperfusion, whereas itincreased Bcl-xl expression and reduced NF-kB in I/R24. CTAP prevented DAMGO effectsin I/R5 and I/R24, showing they are μOR-mediated. Furthermore, DAMGO at concentrationseffective in reducing the inflammatory response, did not affect transit in CTR and SO norin I/R5, whereas it reversed the GI transit delay induced by I/R24. Conclusion: DAMGOreduces early I/R inflammatory response through upregulation of HO-1, an inducible enzymethat acts as defense mechanism against oxidative stress, perhaps via activation of MAPK/ERK signaling. DAMGO beneficial effect on I/R24 is likely to be mediated by suppressionof NF-kB and upregulation of the antiapoptotic factor, Bcl-xl. Finally, DAMGO-inducedreduction of the initial inflammatory response prevented the impairment of GI motility thatoccurred in I/R24.

Su1913

Intracerebroventricular Urocortin 1-Induced Anorexia Involves Peripheral α2Adrenergic Receptor Mediated Inhibition of Ghrelin in Rats: Prevention byRikkunshitoKoji Yakabi, Mitsuko Ochiai, Shoki Ro, Eriko Hosomi, Kenjiro Hayashi, Shino Ohno,Yumi Harada, Tomohisa Hattori, Lixin Wang, Yvette Tache

Background: Corticotropin-releasing factor (CRF) plays a key role in coordinating thehormonal, autonomic, and behavioral responses to stressors. In particular, activation of CRFreceptors suppresses feeding behavior in rodents. Previously we reported that intracerebro-ventricular (ICV)-urocortin 1 (UCN)-induced anorexia is associated with sympatheticmediated inhibition of gastric ghrelin secretion. Rikkunshito (RKT) is one of the Japanesetraditional medicines that increases plasma ghrelin in humans, dogs, and mice. Aim:Toinvestigate the role of peripheral adrenergic receptors (AR) in ICV UCN inhibitory actionsand the influence of RKT. Methods:Adult male fasted rats with chronically implanted ICVcannula were injected with UCN (300 pmol/rat) or phosphate-buffered saline (PBS). RKT(1000 mg/kg/10 mL) was orally administered 1 h before ICV UCN injection. The AR agonistsor antagonists were given intra-peripherally at 0 min or 15 min before ICV UCN or PBS.Cumulative food intake was monitored at 2 h after ICV UCN. Plasma acyl ghrelin levelswere determined 2 h after ICV UCN by ELISA in separate experiments. Binding inhibitoryactivities of RKT components were performed using human recombinant ARs (α2, β) or ratbrain, submaxillary gland, or liver tissues (α1) and radioligand binding assay.Results:RKTprevented (P < 0.05) the inhibition of food intake and decrease ghrelin levels induced byICV UCN. RKT action to restore food intake was abolished by co-administration of theghrelin receptor antagonist ([D-Lys3]-GHRP-6, 4 μmol/kg, intravenously). ICV UCN-induceddecreases in plasma ghrelin levels was not blocked by the selective β1 or β2-AR antagonist(atenolol, 10 mg/kg or ICI118,551, 0.1 mg/kg ) The selective α2-AR antagonist (yohimbine,5 mg/kg) prevented ICV UCN-induced inhibition of ghrelin and food intake (P < 0.05),while the selective α1-AR antagonist (prazosin, 5 mg/kg) had no effect. The α2-AR agonist(clonidine, 5 mg/kg) further enhanced ICV UCN-induced inhibition of ghrelin by 56% (P< 0.01) while the selective α1-AR agonist (phenylephrine, 5 mg/kg) had no effect. Further-more, the selective α2-AR agonist (clonidine, 0.2, 1, and 5 mg/kg) dose-dependentlydecreased acyl ghrelin levels by 43% (P < 0.01), 75% (P < 0.01), and 69% (P < 0.01) innormal fasted rats. The crude components contained in RKT, glycycoumarin (IC50: 5.2 ±0.7 μmol/L), 10-gingerol (IC50: 5.4 ± 0.2 μmol/L), 8- and 6-shogaol (IC50: 24.7 ± 2.4 μmol/L, and 5.7 ± 0.8 μmol/L), and eudesmol, (IC50: 37.2 ± 4.4 μmol/L) inhibited the bindingto α2-AR.Conclusions:1. Activation of peripheral α2-AR inhibits fasting levels of ghrelinand mediated ICV UCN-induced inhibition of acyl ghrelin and related suppression of foodintake; 2. RKT prevents ICV UCN induced anorexia by restoring ghrelin that may involveinteraction with peripheral α2-AR.

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