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H2S Induces Analgesia Is Mediated In Vivo Activation of μ Opioid Receptorand PI3K/AKT Pathway ActivationEleonora Distrutti, Sabrina Cipriani, Barbara Renga, Marco Migliorati, Andrea Mencarelli,Stefano Fiorucci

Background and aim. Hydrogen sulfide (H2S) functions as a neuromodulator and reversescolorectal distension (CRD)-induced pain. Opioid system is widely involved in analgesia.Whether the opioid receptors are involved in the H2S -induced analgesia is unknown.Methods. Four, graded (0.4-1.6 ml water) CRD were produced in awake rats and colorectalpain were assessed by measuring the abdominal withdrawal response (AWR) after i.p.administration of saline or Na2S, a donor of H2S (100 mMol/kg). Spinal cFos expressionwas also detected. To determine whether the opioid system was involved in the H2S -inducedantinociception, pre-treatment with non-selective opioid antagonist naloxone (0.5 mg/kgi.p.) or selective antagonists of μ(MOR) (CTAP, 0.09 mg/kg), k (KOR) (GNTI, 0.08 mg/kg)and δ (DOR) (NTI, 4 mg/kg) opioid receptors or antisense oligodeoxynucleotides directedagainst specific exons of the DOR, KOR and MOR (10 μg each probe/10 μl saline). i.c.v.administered were performed and the AWR score detected. The H2S (50 μM) In Vitro effectsof H2S on MOR phosphorylation, β-arrestins recruitment and MOR internalization weredetermined in the neuronal cell line SKNMC and compared with those obtained with theMOR agonist DAMGO (1 μ). The effects of H2S and DAMGO on PI3K/AKT pathway wasalso detected. Results. H2S significantly inhibited the CRD-induced pain and spinal cFosoverexpression. The H2S-induced analgesia was significantly reverted by naloxone, CTAPand antisense oligodeoxynucleotides against MOR, while both DOR and KOR inhibitionappeared less effective. In the SKNMC cells, both H2S and DAMGO caused the time-dependent MOR phosphorylation on serine 377 and the co-immunoprecipitation betweenMOR and β-arrestin, indicating that H2S induced the interaction of b-arrestins with MOR.Finally, plasma membrane fraction and immunoflurescence staining demonstrated that bothDAMGO and H2S caused the translocation of MOR from the plasma membrane to thecytoplasma and that MOR internalization was inhibited by pre-treating cells with CTAP (1μM). Both DAMGO and H2S induced AKT phosphorylation. CTAP inhibited the DAMGO-induced AKT phosphorylation but not those induced by H2S. Finally, pre-treating neuronalcells with the PI3K inhibitor LY294002 caused the blockade of H2S-induced MOR internaliz-ation. Conclusions. This study provides evidence that: 1) 2Sinhibits CRD-induced pain byinteracting with peripheral and central MOR; 2) H2S causes MOR phosphorylation, theassociation between MOR and β-arrestins and MOR internalization; 3) H2S induces AKTphosphorylation; 4) inhibition of PI3K/AKT pathway results in the blockade of H2S-inducedMOR internalization.

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Synergistic Interaction Between Leptin and CCK in the Nodose Ganglia toMediate Short-Term Satiety Involves JAK/STAT3, SRC, and PI3 KinaseSignaling PathwaysAndrea Heldsinger, Gintautas Grabauskas, Il Song, Chung Owyang

Recent studies show a synergistic interaction between vagal CCK-A receptors (CCKAR) andleptin receptors (LRb) mediates short-term satiety. In fact the satiety action of CCK is entirelydependent on leptin signaling (GE 2008;134:A96). We hypothesize that this synergisticinteraction is mediated by a cross talk between the signaling cascades utilized by CCKARand LRb. Whole cell patch clamp recordings were performed on neurons isolated from ratnodose ganglia (NG). Leptin (10 nM) and CCK-8 (10 nM) each evoked short latency,transient depolarization leading to action potentials in 20% (10/48) and 35% (17/48) ofrecorded neurons respectively. The depolarizing effect of leptin and CCK was associatedwith a 33+2% and 36%+9% increase in membrane input resistance respectively. The I-Vrelationship showed that the current reversed at -100 mV for each peptide, which is closeto the K+ equilibrium potential (-105 mV). Six of 48 neurons responded to both leptin andCCK-8 and showed potentiation between the two peptides, resulting in a 58+12% increasein membrane input resistance. Application of STAT3 inhibitor (10 μM) abolished the actionof leptin and CCK, suggesting phosphorylated STAT3 (pSTAT3) regulates the closure of K+

channels. To identify the signal transduction pathways involved we performed Westernimmunoblotting of pSTAT3 in rat NG. Leptin and CCK-8 each caused a dose (0.1-10 nM)dependent increase in pSTAT3 after 10 min stimulation. A combination of 0.1 nM of leptinand CCK-8 synergistically stimulated a 6.1-7.5 fold increase in pSTAT3. In addition to theJanus-activated kinase (JAK)-STAT pathway, leptin also regulates other key signaling path-ways such as the ERK 1/2 via the MAPK cascades and phosphaticylinositol-3-kinase (PI3K)which is mediated by IRS 1/2. We showed that the synergistic stimulation of pSTAT3 byleptin and CCK-8 was >80% inhibited by the JAK2 inhibitor (AG 490), PP2 (SRC kinaseinhibitor) and the PI3 kinase inhibitors (LY294002 and Wortmannin). However the interac-tion was not affected by PD98059 (MAPK inhibitor). Transient lipofectamine transfectionof cultured NG with siRNA of SRC or PI3 kinase but not MAPK resulted in a loss of leptin/CCK stimulated phosphorylation of STAT3 compared to cells transfected with control siRNA.We conclude that the synergistic interaction between vagal CCKR and LRb is mediated byphosphorylation of STAT3 which in turn activate the closure of K+ channel leading tomembrane depolarization and neuronal firing. This involves the interaction between CCK/SRC cascades and the leptin/JAK/PI3/STAT3 signaling pathways. Malfunctioning of thesesignaling molecules may result in eating disorders.

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Contribution of Na-K-2Cl Cotransporter to Gamma-Amino Butyric Acid(GABAA) Receptor-Mediated Membrane Depolarization in Guinea PigMyenteric NeuronsSumei Liu, Wei Ren, Mei-Hua Qu, Hong Xue, Tuo Ji, Jinxia Zhu, Jackie D. Wood

Background and Aims: The Na-K-2Cl cotransporter (NKCC) elevates neuronal Cl concentra-tion while GABA opens ligand-gated channels that are permeable to Cl. Channel openingmoves the membrane potential toward the Cl equilibrium potential (ECl), which is inhibitory

A-98AGA Abstracts

as long as the threshold for action potential generation is more positive than ECl andexcitatory if ECl is more positive than the action potential threshold. We tested the hypothesisthat NKCC influence on ECl are factors in the excitatory action of GABA in enteric neurons asopposed to inhibitory action of GABA on excitability of CNS neurons. Methods: Intracellular“sharp” electrodes were used to record GABA-evoked responses. Immunofluorescence andWestern blot analysis were used to study expression of NKCC. Results: Application of 1-mM GABA by pressure micro-ejection evoked membrane depolarization associated withdecreased input resistance in 9/10 AH-type and 3/10 S-type neurons. The GABAA antagonist,bicuculline (10μM), suppressed GABA-evoked responses. Bath application of the NKCCinhibitor, bumetanide (100μM), reduced the GABA-evoked depolarizing responses by61.792.91% (control 10.00±1.31 mV vs 3.78±0.46 mV in bumetanide; P<0.001; n=9). Thereversal potential for GABA-evoked depolarization shifted in the hyperpolarizing directionin bumetanide. Application of 300-μM furosemide reduced the GABA-evoked depolarizationby 30.72±6.85% (control 13.50±1.94 mV vs 9.75±2.10 mV in furosemide; P<0.001; n=4).The KCCblocker DIOA (100μM) had no effect on GABA responses ( P>0.05; n=3). Immunob-lots showed expression of NKCC1 (~160 kDa) and NKCC2 (~140 kDa). Immunoreactivity(IR) for NKCC1 and NKCC2 was expressed in both plexuses. Double labeling with antibodiesspecific for the pan neuronal marker HuC/D and a glial cell marker S-100 confirmed thatNKCC1 and NKCC2 IR were expressed by enteric neurons, but not enteric glial cells. BothNKCC1-IR and NKCC2-IR co-localized with the GABAA receptor. Conclusion: The resultssuggest that intraneuronal Cl accumulation by the cation-chloride cotransporter NKCC allowsfor Cl efflux during GABAA receptor activation, thus leading to membrane depolarization.Decreased expression of NKCC and increased expression of KCC in the brain during develop-ment is postulated to be responsible for a switch from excitatory action of GABA in theyoung brain to an inhibitory action in the adult brain. If true, it can be concluded that sucha developmental change might not occur in the ENS. Acknowledgement: Supported by NIHR01 DK37238 and R01 DK57075 (JD Wood), PhRMA Award (S Liu), North Carolina Univ.Center for Functional GI & Motility Disorders seed grant (S Liu).

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Caveolae-Dependent Internalization and Homologous Desensitization of VIP/PACAP Receptor, VPAC2 in Gastrointestinal Smooth MuscleJennifer Kim, Sally Fayed, Sunila Mahavadi, Gracious R. Ross, Syed R. Ahmed,Mohammed Khaleduzzaman, Karnam S. Murthy

The main membrane proteins of caveolae (caveolin-1, -2 and -3) oligomerize within lipid richdomains to form regular invaginations of smooth muscle plasma membrane and participate inreceptor internalization and desensitization independent of clathrin-coated vesicle endo-cytosis. We have previously shown that Gs protein-coupled VIP/PACAP receptors, VPAC2,predominantly expressed in smooth muscle cells of the gut, are exclusively phosphorylatedby GRK2 leading to receptor internalization and desensitization. AIM. To identify the roleof caveolin-1 in VPAC2 receptor internalization and desensitization in gastric smooth muscle.METHODS. The role of caveolin-1 in VPAC2 receptor internalization and desensitizationwas examined using three approaches: 1) methyl cyclodextrin to deplete cholesterol anddisrupt caveolae in dispersed muscle cells, 2) caveolin-1 siRNA to suppress caveolin-1expression in cultured muscle cells, and 3) caveolin-1 knock out mice (caveolin-1-/-). Smoothmuscle cells were isolated from rabbit stomach and from wild type and caveolin-1-/- mice.VPAC2 receptor internalization and desensitization were determined as decrease in [125I]VIPbinding and VIP-induced adenylate cyclase activity, respectively, after treatment with VIPfor 30 min. Desensitization of the response was determined by decrease in VIP-inducedmuscle relaxation in both isolated muscle cells and muscle strips after treatment with VIPfor 30 min. Relaxation was measured by scanning micrometry in isolated muscle cells andisometrically with a force-displacement transducer in muscle strips. RESULTS. In bothfreshly dispersed and cultured muscle cells of rabbit stomach, pretreatment with VIP causedinternalization of VPAC2 receptors (70%-76% decrease in binding) and desensitization ofVIP-induced adenylate cyclase activity (65%-70% decrease in VIP-induced cAMP formation).Internalization and desensitization were blocked by either disruption of caveolae with methylcyclodextrin in freshly dispersed muscle cells or suppression of caveolin-1 with caveolin-1siRNA in cultured muscle cells. VIP caused relaxation in isolated muscle cells and musclestrips. Pretreatment with VIP significantly inhibited relaxation in response to subsequentaddition of VIP in freshly dispersed muscle cells and in muscle strips isolated from wildtype mice, but not from caveolin-1-/- mice. These results suggest that caveolin-1 plays animportant role in VPAC2 receptor internalization and desensitization. CONCLUSION. Ingastrointestinal smooth muscle, VPAC2 receptor internalization and desensitization are medi-ated via caveolar pathway.

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Upregulation of Vagal NK1 Receptor By Cisplatin Facilitates the Interaction ofSubstance P and Serotonin in the Vagal Afferent Neurons: Implication in theEmesis Related to ChemotherapyXiaoyin Wu, Jing Fan, Chung Owyang, Ying Li

Anti-cancer chemotherapy such as cisplatin often induces emesis. This action has beenattributed to the release of serotonin (5-HT) from intestinal EC cells which act on vagal5HT3 receptors. Recently NK-1 receptor antagonists have shown some efficacy in preventingdelayed emesis. Since EC cells also contain substance P (SP) and NK1 receptors are presentin nodose ganglia (NG), we hypothesize that cisplatin causes delayed emesis through theinteraction of 5HT3 and NK-1 signaling pathways on vagal afferent neurons. To examinethis hypothesis, we investigated the actions of 5-HT and SP on vagal afferent firings andexamined the plasticity of vagal afferent neurons after cisplatin. Single vagal afferent neuronaldischarges were recorded In Vivo in rats. Intra-arterial injection of SP at 5 and 10 μg/kgproduced a dose-dependent increase in firing rates in 12/78 neurons (0.5±0.2 at basal to11±3 and 37±6 impulses/20 s, respectively). 7/12 of the SP-sensitive neurons were alsoactivated by luminal perfusion of 5-HT (10-5M, 20 ± 3 impulses/20 s). To investigate theinteraction between 5-HT and SP, 21/98 neurons were activated by SP and 8 of these 21neurons also responded to 5-HT (10-5M). A subthreshold dose of 5-HT (10-7M) producedno measurable electrophysiological effects but augmented the neuronal responses to 5, 10