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Chronic Stress-Induced Visceral Hyperalgesia: Evidence for EpigeneticRegulation of DNA Methyltransferase I (Dnmt1) in Dorsal Root Ganglion(DRG) Neurons Innervating the Colon in the RatShuangsong Hong, Gen Zheng, John W. Wiley

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifiesepigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed byDNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptionalcontrol of gene expression. We reported previously that chronic psychological stress inducedvisceral hyperalgesia and differential alterations in the expression of several genes in DRGneurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheralsensory pathways has not been investigated. Objectives: We examined the hypothesis thatDNMTs play an important role in the regulation of chronic stress-induced visceral hyperalge-sia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situto L6-S2 DRGs every other day during the stress procedure. The visceromotor response(VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B(CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWesternblot analysis were used to assess protein expression. In Vitro studies were performed inisolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) andRU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstratedsignificant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGscompared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significantincrease in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) com-pared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% ofthe CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed ratscompared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats wasincreased 68% and 92% above control responses at pressures of 40 and 60 mm Hg,respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhance-ment and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment ofcontrol L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level thatwas prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the noveland provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genesin primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an importantrole in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltrans-ferases represent a potential target for treatment of functional GI disorders associated withvisceral hyperalgesia.

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Identification of Colonic Afferent Central Terminals and Changes FollowingColonic InflammationAndrea Harrington, Stuart M. Brierley, Joel Castro, Patrick A. Hughes, L. AshleyBlackshaw

Background: Peripheral endings of mechanosensitive colonic afferent neurons are well charac-terized [1] and become mechanically hypersensitive following inflammation [2]. As yet, littleis known about their central terminals located in the spinal cord. We therefore aimed toidentify colonic afferent central terminals and the dorsal horn (DH) neurons activated bycolonic distension (CRD). Furthermore, as sensitization of spinal cord nociceptive pathwayscommonly follows peripheral hypersensitivity, causing altered and referred pain perception,we aimed to identify changes in colonic afferent central terminals after colitis. Methods:Experiments were performed in healthy mice (n=4) and a model of post-inflammatorychronic colonic hypersensitivity (n=4). TNBS (130μL/ml) was administered by colorectalenema and mice were allowed to recover for 28 days before fluorescent retrograde tracerwas injected into the colon wall. Mice underwent CRD (80mmHg), followed by fixationand removal of spinal cord (T10-L1) for immunohistochemistry. Labelling for calcitoningene related peptide (CGRP) and isolectin B4 (IB4) identified DH layers. Labelling forphosphorylated MAP kinase ERK 1/2 (pERK) identified DH neurons activated by CRD. Thenumber of pERK-immunoreactive (IR) cells was compared between healthy and post-inflamedmice using unpaired student t-tests. Results: Traced colonic afferent central terminals werepresent in CGRP-IR positive/IB4-IR negative layers of the spinal cord DH and projecteddown mid and lateral DH collateral pathways into lamina V. In post-inflamed mice terminalswere denser in CGRP-IR DH laminae and now projected into IB4-IR layers and deeper DHlaminae. In healthy mice, CRD evoked pERK-IR in 71.5±17 DH neurons, most of whichwere located in DH lamina I and closely apposed to labeled colonic afferent terminals. Inpost-inflamed mice, the number of pERK-IR neurons following CRD significantly increased(105±11.7, p<0.05), with the greatest increase (78.5±13 to 199±9; p<0.005) occurring inT13-L1. pERK-IR neurons were now located in DH laminae I and II and scattered throughoutdeeper laminae, where populations were closely apposed to colonic afferent terminals.Conclusion: Colonic afferent central terminals project onto spinal cord DH neurons activatedby colonic distension. In post-inflamed mechanically hypersensitive mice, increases in projec-tions were observed into different DH laminae, which correlated with additional DH neuronsstimulated by CRD. Such changes may facilitate enhanced signalling of mechanical stimuliresulting in sensitization of central nociceptive pathways and chronic mechanical allodynia.How the neuropharmacology of colonic afferent central terminals and spinal targets areaffected by colonic inflammation is being determined. 1)Brierley et al., Gastroenterology.2004; 2) Hughes & Brierley, et al. Gut. 2009.

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776

Cytokine Modulation of Visceral Afferents via Cation Channels is Switched inChronic Visceral HypersensitivityPatrick A. Hughes, Andrea Harrington, Stuart M. Brierley, Lochana Maldeniya, ChrisMartin, Jenny Persson, Tobias Liebregts, Birgit Adam, Gerald Holtmann, Jane M.Andrews, L. Ashley Blackshaw

The contribution of the immune system to the symptoms of Irritable Bowel Syndrome (IBS)is currently controversial. Peripheral Blood Mononuclear cell (PBMC) supernatants sensitisecolonic afferents to mechanical stimuli and have increased concentrations of IL-6 andTNFα1,2.We have developed amousemodel of post inflammatory chronic visceral hypersens-itivity (CVH) whereby colonic afferents are mechanically hypersensitive3. Aim: To understandthe mechanisms underlying the effects of inflammatory mediators on colonic afferent mech-anosensitivity in health and CVH. Methods: PBMCs were isolated from whole blood ofpost inflammatory / diarrhoea predominant (PI/D) IBS patients and supernatants collected.Afferent mechanosensitivity was assessed in mouse colo-rectum In Vitro with afferents charac-terised by mechanical threshold. Changes in afferent mechanosensitivity were assessed inresponse to PBMC supernatants, cytokines or inhibitors of ion channels. Cytokine receptorexpression was assessed by quantitative RT-PCR of retrogradely labelled laser capture microd-issected colonic DRG neurons. Comparisons were made between healthy and CVH (TNBS130μg/mL with 28 day recovery) mice. Results: In healthy mice PBMC supernatants fromPI/D IBS patients sensitised colonic afferents to mechanical stimuli (P<0.05, n=6). Theseeffects were mimicked by 100ng/mL IL-6 (P<0.05, n=6) and TNFα (P<0.001, n=10). Prein-cubation with the selective TRPA1 antagonist (HC-030031 10μM) abolished the sensitisingeffects of IL-6 and TNFα, whereas inhibition of TRPV1 (capsazepine 500μM) or NaV(tetrodotoxin 1μM) channels had no effect. Surprisingly in CVH a switch in the functionaleffect toward inhibition of mechanosensitivity was observed for supernatants (n=7), and forboth IL-6 (P<0.05, n=5) and TNFα (P<0.001, n=7). Inhibition by IL-6 and TNFα wasabolished by blocking the BKCa potassium channel (iberiotoxin 100μM). Colonic DRGneurons of CVH mice had increased mRNA expression of TNFR1 (P<0.05) and BKCa(P<0.01), while IL-6R and TRPA1 were maintained and the IL-6 co-receptor CD130 wasreduced (P<0.05). TNFR2 was not expressed in colonic DRG neurons in healthy or CVHmice. Conclusions: In health the cytokines IL-6 and TNFα sensitise colonic afferents viacoupling of their receptors with TRPA1. However in CVH a switch occurs in both caseswhereby these cytokines now couple with the inhibitory BKCa channel to reduce mechano-sensitivity. These results indicate these cytokines share common mechanisms of action inhealth and post-inflammatory settings. In the latter, BKCa channels may be recruited tomitigate excitotoxity via othermechanisms. Amore detailed understanding of themechanismsunderlying the switch in effect may reveal targets for treatment of post inflammatory pain,such as PI IBS. 1: Hughes et. al. Gut 2009. 2: Liebregts et. al. Gastro. 2007. 3: Hughes et.al. Gut 2009

777

Effect of Prostaglandin D2 on Vagal Afferent C-Fibers in Guinea PigEsophagusShaoyong Yu

Our previous studies demonstrated that mast cell activation led to sensitization of esophagealnodose C-fibers, which involved certain mast cell preformed mediators. Mast cell activationalso releases de novo synthesized lipid mediators including prostaglandin D2 (PGD2) andleukotrienes. The effect of PGD2 on esophageal vagal afferents has yet to be determined.Aim: to determine the effect of PGD2 on esophageal vagal afferent C fibers. Methods: Extra-cellular single fiber recordings were performed from vagal nodose neurons using ex vivoesophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilitieswere compared by action potentials evoked by esophageal distensions before and afterperfusion of PGD2 with/without pretreatment of PGD2 DP receptor antagonist BWA 868C.Results: PGD2 treatment (10μM, 30-min) either had no (n=8) or trivial (<1 Hz, n=4) effectat overtly causing action potential discharge in the vagal nodose C-fibers. By contrast, PGD2treatment significantly and substantively enhanced C-fiber responses to esophageal distension(p < 0.01). The peaks of action potential discharges per 20 second stimulation at esophagealdistension pressures of 10, 30 and 60 mmHg averaged 3.6±0.4, 7.7±0.6 and 11.9±0.7 Hz,respectively. This response was increased 2 fold by PGD2 perfusion for 30-min (7.9 ±0.8;15.4±1.1, and 21.5±1 Hz, respectively, p < 0.01, n=12). The increased responses sustainedfor more than 30-min after washing PGD2 out (with the peaks of action potentials averaged8.0 ±0.9; 14.8±1.5, and 22.4±1.8 Hz, respectively; vs control, p < 0.01, n=10). Pre-treatmentwith PGD2 DP receptor antagonist BWA 868C (10μM, 30-min) prevented PGD2-inducedincreases in mechano-excitability in nodose C fibers. Esophageal distension-evoked actionpotentials did not significantly change after PGD2 perfusion following 30-min pretreatmentand continually in the presence of BWA 868C (control vs PGD2+BWA868C: 2.5 ±0.3;4.8±0.5, 8.1±0.7 Hz vs 2.9 ±0.4; 5.5±0.6, 9.5±0.9 Hz, respectively, p > 0.05, n=10) Conclu-sion: our data provides the first evidence directly from visceral sensory nerve endings thatPGD2 increasesmechano-excitability of esophageal nodose C-fibers, which initiated via PGD2DP receptor. This reveals a novel mechanism mediating visceral peripheral sensitization.Supported by NIH grant DK087991.

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Regulation of CUG-binding Protein 1 Translation Through MicroRNA-503 byPolyamines Modulating Normal Intestinal Mucosal GrowthYu-Hong Cui, Lan Xiao, Jaladanki N. Rao, Tongtong Zou, Lan Liu, Alexis D. Smith, Jian-Ying Wang

Polyamines are required for normal gut mucosal growth by regulating expression of variousgenes, but a little is known about the exact roles of polyamines at the molecular level.Recently, polyamines are shown to regulate expression and subcellular trafficking of RNA-binding proteins (RBPs) such as CUB-binding protein 1 (CUGBP1) and HuR, thus modulatingthe stability and translation of several mRNAs encoding growth-regulatory proteins. However,

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