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1.
Artigo em Inglês | MEDLINE | ID: mdl-28799255

RESUMO

BACKGROUND: Gastroprokinetic properties of 5-HT4 receptor agonists, such as prucalopride, are attributed to activation of 5-HT4 receptors on cholinergic nerves innervating smooth muscle in the gastrointestinal smooth muscle layer, increasing acetylcholine release and muscle contraction. In porcine stomach and colon, phosphodiesterase (PDE) 4 has been shown to control the signaling pathway of these 5-HT4 receptors. The aim of this study was to investigate the PDE-mediated control of these 5-HT4 receptors in human large intestine. METHODS: Circular smooth muscle strips were prepared from human large intestine; after incubation with [³H]-choline, electrically induced tritium outflow was determined as a measure for acetylcholine release. The influence of PDE inhibition on the facilitating effect of prucalopride on electrically induced acetylcholine release was studied. KEY RESULTS: The non-selective PDE inhibitor IBMX enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence, while rolipram and roflumilast (PDE4) enhanced the prucalopride-induced facilitation to the same extent as IBMX. CONCLUSIONS & INFERENCES: In human large intestinal circular muscle, the intracellular pathway of 5-HT4 receptors facilitating cholinergic neurotransmission to large intestinal circular smooth muscle is controlled by PDE4. If the synergy between 5-HT4 receptor agonism and PDE4 inhibition is confirmed in a functional assay with electrically induced cholinergic contractions of human large intestinal circular smooth muscle strips, combination of a selective 5-HT4 receptor agonist with a selective PDE4 inhibitor might enhance the in vivo prokinetic effect of the 5-HT4 receptor agonist in the large intestine.


Assuntos
Acetilcolina/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Intestino Grosso/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , 1-Metil-3-Isobutilxantina/administração & dosagem , Idoso , Benzofuranos/administração & dosagem , Feminino , Humanos , Intestino Grosso/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Inibidores de Fosfodiesterase/administração & dosagem , Agonistas do Receptor 5-HT4 de Serotonina/administração & dosagem
2.
Artigo em Inglês | MEDLINE | ID: mdl-28332745

RESUMO

BACKGROUND: In the gastrointestinal tract of several species, facilitating 5-HT4 receptors were proposed on myenteric cholinergic neurons innervating smooth muscle by in vitro study of the effect of the selective 5-HT4 receptor agonist prucalopride on submaximal cholinergic contractions. This was not yet established in the murine gastrointestinal tract. METHODS: In circular smooth muscle strips from murine fundus, jejunum and colon, contractions were induced by electrical field stimulation in the presence of guanethidine, L-NAME and for colon also MRS 2500. Submaximal contractions were induced to study the influence of prucalopride. KEY RESULTS: Electrical field stimulation at reduced voltage induced reproducible submaximal neurogenic and cholinergic contractions as the contractions were abolished by tetrodotoxin and atropine. Hexamethonium had no systematic inhibitory effect but mecamylamine reduced the responses, suggesting that part of the cholinergic response is due to activation of preganglionic neurons. Prucalopride concentration-dependently increased the submaximal cholinergic contractions in the three tissue types, reaching maximum from 0.03 µmol/L onwards. The facilitation in the different series with 0.03 µmol/L prucalopride ranged from 41% to 104%, 30% to 76% and 24% to 74% in fundus, jejunum, and colon, respectively. The effect of 0.03 µmol/L prucalopride was concentration-dependently inhibited by GR 113808. CONCLUSIONS & INFERENCES: In the murine gastrointestinal tract, activation of 5-HT4 receptors with prucalopride enhances cholinergic contractions, illustrating facilitation of myenteric cholinergic neurotransmission. The degree of enhancement with prucalopride is of similar magnitude as previously reported in other species, but the effective concentrations are lower than those needed in the gastrointestinal tract of other species.


Assuntos
Acetilcolina/fisiologia , Trato Gastrointestinal/fisiologia , Receptores 5-HT4 de Serotonina/fisiologia , Transmissão Sináptica , Animais , Benzofuranos/administração & dosagem , Colo/efeitos dos fármacos , Colo/fisiologia , Nucleotídeos de Desoxiadenina/administração & dosagem , Estimulação Elétrica , Fundo Gástrico/efeitos dos fármacos , Fundo Gástrico/fisiologia , Trato Gastrointestinal/efeitos dos fármacos , Guanetidina/administração & dosagem , Hexametônio/administração & dosagem , Jejuno/efeitos dos fármacos , Jejuno/fisiologia , Masculino , Mecamilamina/administração & dosagem , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , NG-Nitroarginina Metil Éster/administração & dosagem , Agonistas do Receptor 5-HT4 de Serotonina/administração & dosagem
3.
Equine Vet J ; 47 Suppl 48: 7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26376095

RESUMO

REASONS FOR PERFORMING STUDY: Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. OBJECTIVES: To investigate whether functional 5-HT4 receptors are present in the equine small intestine. STUDY DESIGN AND METHODS: In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. RESULTS: Results were similar in both muscle layers. In the presence of 0.3 mmol/l NG-Nitro-L-arginine methyl ester and 0.3 µmol/l apamine, excluding effects of the inhibitory transmitters NO and ATP, EFS induced voltage-dependent on-contractions; these were neurogenic as they were abolished by 3 µmol/l tetrodotoxin. At a voltage inducing 50% of the maximal amplitude, the submaximal EFS-induced contractions were cholinergic as atropine (1 µmol/l) abolished them. Prucalopride (0.3 µmol/l) did not increase the amplitude of these submaximal EFS-induced contractions. Even in the presence of the nonselective phosphodiesterase inhibitor IBMX, previously shown to enhance the effect of neuronal 5-HT4 receptors by inhibiting breakdown of their 2nd messenger cAMP [5], prucalopride (3 µmol/l) had no influence. Also 5-HT (10 µmol/l), a full agonist at 5-HT4 receptors, tested in the presence of methysergide and granisetron to exclude interaction with other 5-HT receptor subtypes, did not enhance EFS-induced submaximal contractions. CONCLUSIONS: There is no evidence for presence of 5-HT4 receptors on the cholinergic neurons of the equine small intestine. These results question the application of 5-HT4 prokinetic drugs in horses. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected at an abattoir. Sources of funding: None. Competing interests: None declared.

4.
Br J Pharmacol ; 172(20): 4864-74, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26227770

RESUMO

BACKGROUND AND PURPOSE: As the pathogenesis of postoperative ileus (POI) involves inflammation and oxidative stress, comparable to ischaemia/reperfusion injury which can be ameliorated with nitrite, we investigated whether nitrite can protect against POI and explored the mechanisms involved. EXPERIMENTAL APPROACH: We used intestinal manipulation (IM) of the small intestine to induce POI in C57BL/6J mice. Sodium nitrite (48 nmol) was administered intravenously just before IM. Intestinal transit was assessed using fluorescent imaging. Bethanechol-stimulated jejunal circular muscle contractions were measured in organ baths. Inflammatory parameters, neutrophil infiltration, inducible NOS (iNOS) activity, reactive oxygen species (ROS) levels, mitochondrial complex I activity and cGMP were measured in the intestinal muscularis. KEY RESULTS: Pre-treatment with nitrite markedly improved the delay in intestinal transit and restored the reduced intestinal contractility observed 24 h following IM. This was accompanied by reduced protein levels of TNF-α, IL-6 and the chemokine CCL2, along with reduced iNOS activity and ROS levels. The associated neutrophil influx at 24 h was not influenced by nitrite. IM reduced mitochondrial complex I activity and cGMP levels; treatment with nitrite increased cGMP levels. Pre-treatment with the NO scavenger carboxy-PTIO or the soluble guanylyl cyclase inhibitor ODQ abolished nitrite-induced protective effects. CONCLUSIONS AND IMPLICATIONS: Exogenous nitrite deserves further investigation as a possible treatment for POI. Nitrite-induced protection of POI in mice was dependent on NO and this effect was not related to inhibition of mitochondrial complex I, but did involve activation of soluble guanylyl cyclase.


Assuntos
Íleus/tratamento farmacológico , Complicações Pós-Operatórias/tratamento farmacológico , Nitrito de Sódio/uso terapêutico , Animais , Quimiocina CCL2/metabolismo , GMP Cíclico/metabolismo , Trânsito Gastrointestinal/efeitos dos fármacos , Íleus/imunologia , Íleus/metabolismo , Íleus/fisiopatologia , Interleucina-6/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Neutrófilos/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Nitrito de Sódio/sangue , Nitrito de Sódio/farmacocinética , Nitrito de Sódio/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
5.
Br J Pharmacol ; 172(6): 1557-73, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24641722

RESUMO

Carbon monoxide (CO) is enzymatically generated in mammalian cells alongside the liberation of iron and the production of biliverdin and bilirubin. This occurs during the degradation of haem by haem oxygenase (HO) enzymes, a class of ubiquitous proteins consisting of constitutive and inducible isoforms. The constitutive HO2 is present in the gastrointestinal tract in neurons and interstitial cells of Cajal and CO released from these cells might contribute to intestinal inhibitory neurotransmission and/or to the control of intestinal smooth muscle cell membrane potential. On the other hand, increased expression of the inducible HO1 is now recognized as a beneficial response to oxidative stress and inflammation. Among the products of haem metabolism, CO appears to contribute primarily to the antioxidant and anti-inflammatory effects of the HO1 pathway explaining the studies conducted to exploit CO as a possible therapeutic agent. This article reviews the effects and, as far as known today, the mechanism(s) of action of CO administered either as CO gas or via CO-releasing molecules in acute gastrointestinal inflammation. We provide here a comprehensive overview on the effect of CO in experimental in vivo models of post-operative ileus, intestinal injury during sepsis and necrotizing enterocolitis. In addition, we will analyse the in vitro data obtained so far on the effect of CO on intestinal epithelial cell lines exposed to cytokines, considering the important role of the intestinal mucosa in the pathology of gastrointestinal inflammation.


Assuntos
Monóxido de Carbono/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Inflamação/patologia , Doença Aguda , Animais , Citocinas/metabolismo , Gastroenteropatias/patologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Humanos , Mucosa Intestinal/patologia , Estresse Oxidativo/fisiologia
6.
Cell Signal ; 26(11): 2573-82, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25101859

RESUMO

Phosphodiesterase (PDE) 3 and PDE4, which degrade cyclic adenosine monophosphate (cAMP), are important regulators of 5-hydroxytryptamine (5-HT) 4 receptor signaling in cardiac tissue. Therefore, we investigated whether they interact with the 5-HT4(b) receptor, and whether A-kinase anchoring proteins (AKAPs), scaffolding proteins that bind to the regulatory subunit of protein kinase A (PKA) and contribute to the spacial-temporal control of cAMP signaling, are involved in the regulation of 5-HT4(b) receptor signaling. By measuring PKA activity in the absence and presence of PDE3 and PDE4 inhibitiors, we found that constitutive signaling of the overexpressed HA-tagged 5-HT4(b) receptor in HEK293 cells is regulated predominantly by PDE4, with a secondary role for PDE3 that is unmasked in the presence of PDE4 inhibition. Overexpressed PDE4D3 and PDE3A1, and to a smaller extent PDE4D5 co-immunoprecipitate constitutively with the 5-HT4(b) receptor. PDE activity measurements in immunoprecipitates of the 5-HT4(b) receptor confirm the association of PDE4D3 with the receptor and provide evidence that the activity of this PDE may be increased upon receptor stimulation with 5-HT. A possible involvement of AKAPs in 5-HT4(b) receptor signaling was uncovered in experiments using the St-Ht31 inhibitor peptide, which disrupts the interaction of AKAPs with PKA. However, St-Ht31 did not influence 5-HT4(b) receptor-stimulated PKA activity, and endogenous AKAP79 and gravin were not found in immunoprecipitates of the 5-HT4(b) receptor. In conclusion, we found that both PDE3A1 and PDE4D3 are integrated into complexes that contain the 5-HT4(b) receptor and may thereby regulate 5-HT4(b) receptor-mediated signaling.


Assuntos
AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Células HEK293 , Humanos , Receptores 5-HT4 de Serotonina/genética
7.
Neurogastroenterol Motil ; 25(5): e339-52, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23551931

RESUMO

BACKGROUND: Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme-free status of sGC, as induced by oxidative stress, was investigated. METHODS: sGCß1 (H105F) knock-in (apo-sGC) mice, which express heme-free sGC that has basal activity, but cannot be stimulated by NO, were generated. KEY RESULTS: Diethylenetriamine NONOate did not increase sGC activity in gastrointestinal tissue of apo-sGC mice. Exogenous NO did not induce relaxation in fundic, jejunal and colonic strips, and pyloric rings of apo-sGC mice. The stomach was enlarged in apo-sGC mice with hypertrophy of the muscularis externa of the fundus and pylorus. In addition, gastric emptying and intestinal transit were delayed and whole-gut transit time was increased in the apo-sGC mice, while distal colonic transit time was maintained. The nitrergic relaxant responses to electrical field stimulation at 1-4 Hz were abolished in fundic and jejunal strips from apo-sGC mice, but in pyloric rings and colonic strips, only the response at 1 Hz was abolished, indicating the contribution of other transmitters than NO. CONCLUSIONS & INFERENCES: The results indicate that the gastrointestinal consequences of switching from a native sGC to a heme-free sGC, which cannot be stimulated by NO, are most pronounced at the level of the stomach establishing a pivotal role of the activation of sGC by NO in normal gastric functioning. In addition, delayed intestinal transit was observed, indicating that nitrergic activation of sGC also plays a role in the lower gastrointestinal tract.


Assuntos
Motilidade Gastrointestinal/fisiologia , Gastroparesia/metabolismo , Guanilato Ciclase/metabolismo , Heme/deficiência , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Modelos Animais de Doenças , Mucosa Gástrica/metabolismo , Técnicas de Introdução de Genes , Guanilato Ciclase/química , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Estresse Oxidativo/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Guanilil Ciclase Solúvel
8.
Naunyn Schmiedebergs Arch Pharmacol ; 385(6): 565-77, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22426996

RESUMO

We studied how 5-HT(4) receptor-mediated inotropic responses are regulated at the level of cAMP in porcine left atrium. We used selective phosphodiesterase (PDE) inhibitors to assess which PDE subtypes are responsible for the fade with time of inotropic responses to 5-HT(4) receptor activation with 5-HT and the 5-HT(4) receptor agonist prucalopride. A possible cross-talk via PDEs between cGMP and 5-HT(4) receptor-induced cAMP signalling was evaluated. Electrically paced left atrial pectinate muscles from young male pigs (15-25 kg) were studied in vitro. Simultaneous inhibition of PDE3 plus PDE4 subtypes was necessary to increase the amplitude and completely prevent the fade of the inotropic response to 5-HT and prucalopride. When responses to 5-HT or prucalopride had faded 1 h after addition, the nonspecific PDE-inhibitor IBMX still fully recovered inotropic responses. Stimulation of particulate guanylyl cyclase, together with PDE2 and PDE4 inhibition, delayed the fade of the response to 5-HT, while stimulation of soluble guanylyl cyclase independently of PDEs accelerated the fade of the response to 5-HT. In conclusion, both PDE3 and PDE4 subtypes are responsible for the suppression and the fade of the inotropic response to 5-HT and prucalopride. Signalling through the 5-HT(4) receptor remains fully active for at least 90 min with PDEs continuously regulating the response. cGMP levels, elevated by activation of particulate guanylyl cyclase under PDE2 inhibition, can indirectly enhance 5-HT(4) receptor-mediated signalling, at least when also PDE4 is inhibited, presumably through inhibition of PDE3. Elevation of cGMP generated by soluble guanylyl cyclase attenuates responses to 5-HT independently of PDEs.


Assuntos
Função Atrial/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Receptores 5-HT4 de Serotonina/fisiologia , Animais , Benzofuranos/farmacologia , AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Guanilato Ciclase/fisiologia , Átrios do Coração/efeitos dos fármacos , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Peptídeo Natriurético Tipo C/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Serotonina/farmacologia , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Guanilil Ciclase Solúvel , Suínos
9.
Neurogastroenterol Motil ; 22(5): 557-63, e121-2, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20025676

RESUMO

BACKGROUND: The 5-HT(4) receptor agonist prucalopride enhances large intestinal contractility by facilitating acetylcholine release through activation of 5-HT(4) receptors on cholinergic nerves and is effective in patients with constipation. Patients with intestinal endometriosis can present with constipation. We investigated in vitro whether large intestinal endometriotic infiltration influences contractility and facilitation of acetylcholine release by prucalopride. METHODS: Sigmoid colon or rectum circular muscle strips were obtained at the level of an endometriotic nodule with infiltration of the Auerbach plexus, and at a macroscopically healthy site at least 5 cm cranially from the nodule, in patients undergoing laparoscopic colorectal resection because of symptomatic bowel endometriosis. Responses to muscarinic receptor stimulation and to electrical field stimulation (EFS), and the facilitating effect of prucalopride on acetylcholine release were evaluated. KEY RESULTS: The EC50 and E(max) of the contractile responses to the muscarinic receptor agonist carbachol did not differ between healthy and lesioned strips. EFS-induced on-contractions were not different between the healthy and lesioned strips, while the non-nitrergic relaxant responses induced by EFS were decreased in the lesioned strips. The facilitating effect of prucalopride on acetylcholine release in healthy strips was similar to that reported before in macroscopically healthy colon tissue of patients with colon cancer; in lesioned strips, the effect of prucalopride was fully maintained in 6/8 patients and absent in two. CONCLUSIONS & INFERENCES: Large intestinal endometriosis does not lead to a systematic interference with the cholinergic facilitating effect of prucalopride.


Assuntos
Acetilcolina/metabolismo , Endometriose/metabolismo , Enteropatias/metabolismo , Intestino Grosso/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Adulto , Área Sob a Curva , Benzofuranos/farmacologia , Estimulação Elétrica , Feminino , Humanos , Intestino Grosso/efeitos dos fármacos , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Processamento de Sinais Assistido por Computador
10.
Br J Pharmacol ; 156(2): 362-76, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19154432

RESUMO

BACKGROUND AND PURPOSE: The time dependency of the effect of 5-HT(4) receptor agonists depends on many specific regulatory mechanisms, which vary between tissues. This has important implications with regard to the effects of endogenous 5-HT, as well as to the clinical use of 5-HT(4) receptor agonists, and might contribute to tissue selectivity of agonists. EXPERIMENTAL APPROACH: The progression and desensitization of 5-HT(4) receptor-mediated responses were evaluated in an organ bath set-up using two, clinically relevant, porcine in vitro models: gastric cholinergic neurotransmission and atrial contractility. KEY RESULTS: Exposure of gastric tissue to 5-HT or to the selective 5-HT(4) receptor agonists prucalopride and M0003 results in a sustained non-transient effect during exposure; after washout, the response to a subsequent challenge with 5-HT shows no clear desensitization. Incubation of left atrial tissue with 5-HT resulted in a transient response, leading after washout to a marked desensitization of the subsequent response to 5-HT. The selective 5-HT(4) receptor agonists prucalopride and M0003 induce only very weak atrial responses whereas they are very effective in desensitizing the atrial response to 5-HT. The observations also suggest that the properties of prucalopride and M0003 to bind to and/or activate the 5-HT(4) receptor differ from those of 5-HT. This difference might have contributed to the observed desensitization. CONCLUSIONS AND IMPLICATIONS: The high potency of prucalopride and M0003 in desensitizing the response to 5-HT together with their low efficacy in the atrium emphasizes the cardiac safety of this class of 5-HT(4) receptor agonists.


Assuntos
Benzofuranos/farmacologia , Músculo Liso/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Agonistas do Receptor 5-HT4 de Serotonina , Estômago/efeitos dos fármacos , Animais , Função do Átrio Esquerdo/efeitos dos fármacos , Benzofuranos/efeitos adversos , Feminino , Técnicas In Vitro , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Especificidade de Órgãos , Receptores 5-HT4 de Serotonina/fisiologia , Antagonistas do Receptor 5-HT4 de Serotonina , Estômago/inervação , Estômago/fisiologia , Suínos , Transmissão Sináptica , Taquifilaxia
11.
Gut ; 58(3): 347-56, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19022916

RESUMO

BACKGROUND AND AIMS: Treatment with carbon monoxide (CO) inhalation has been shown to ameliorate postoperative ileus (POI) in rodents and swine. The aim of this study was to investigate whether CO liberated from water-soluble CO-releasing molecules (CO-RMs) can protect against POI in mice and to elucidate the mechanisms involved. METHODS: Ileus was induced by surgical manipulation of the small intestine (IM). Intestinal contractility-transit was evaluated by video-fluorescence imaging. Leucocyte infiltration (myeloperoxidase), inflammatory parameters (ELISA), oxidative stress (lipid peroxidation), and haem oxygenase (HO)/inducible nitric oxide synthase (iNOS) enzyme activity were measured in the intestinal mucosa and muscularis propria. RESULTS: Intestinal contractility and transit were markedly restored when manipulated mice were pre-treated with CO-RMs. Intestinal leucocyte infiltration, expression levels of interleukin 6 (IL6), monocyte chemoattractant protein-1 and intercellular adhesion molecule-1, as well as iNOS activity were reduced by treatment with CORM-3 (a transition metal carbonyl that releases CO very rapidly); whereas expression of IL10/HO-1 was further increased when compared to nontreated manipulated mice. Moreover, treatment with CORM-3 markedly reduced oxidative stress and extracellular signal-related kinase (ERK)1/2 activation in both mucosa (early response) and muscularis (biphasic response). The p38 mitogen-activated protein kinase inhibitor SB203580 abolished CORM-3-mediated HO-1 induction. The HO inhibitor chromium mesoporphyrin only partially reversed the protective effects of CORM-3 on inflammation/oxidative stress in the muscularis, but completely abrogated CORM-3-mediated inhibition of the early "oxidative burst" in the mucosa. CONCLUSIONS: Pre-treatment with CO-RMs markedly reduced IM-induced intestinal muscularis inflammation. These protective effects are, at least in part, mediated through induction of HO-1, in a p38-dependent manner, as well as reduction of ERK1/2 activation. In addition, CORM-induced HO-1 induction reduces the early "oxidative burst" in the mucosa following IM.


Assuntos
Monóxido de Carbono/administração & dosagem , Heme Oxigenase-1/biossíntese , Íleus/prevenção & controle , Intestino Delgado , Estresse Oxidativo/efeitos dos fármacos , Complicações Pós-Operatórias/prevenção & controle , Administração por Inalação , Animais , Carboxihemoglobina/metabolismo , Trânsito Gastrointestinal/fisiologia , Heme Oxigenase-1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Distribuição Aleatória , Transdução de Sinais
12.
Equine Vet J ; 40(4): 326-31, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18321805

RESUMO

REASONS FOR PERFORMING STUDY: Increased plasma (5-HT) concentrations are reported in horses predisposed to develop laminitis and after i.v. infusion of endotoxins. In the equine jejunum contractile 5-HT1A-like receptors show tachyphylaxia upon prolonged activation with 5-HT. Therefore, increased systemic 5-HT release in colic horses could play a possible role in the pathophysiology of ileus. OBJECTIVE: To investigate possible increased systemic release of 5-HT in colic horses with compromised bowel and to identify the source of 5-HT overload. METHODS: Concentrations of 5-HT were determined in plasma and peritoneal fluid (PF) of healthy horses (n = 10), strangulating small intestinal colic horses (n = 18), nonsurgical colic horses (n = 10) and cryptorchid stallions (n = 6). It was attempted to identify the source of 5-HT overload by comparing the blood and PF 5-HT concentrations within horses and by assessing the in vivo platelet activation through determination of the beta-thromboglobulin (beta-TG)/platelet factor 4 (PF4) ratio. RESULTS: All horses in the strangulating small intestinal colic group had plasma (P = 0.006) and PF (P = 0.01) 5-HT concentrations above those found in the control group. Plasma beta-TG/PF4 ratio in these horses exceeded 2 in all cases, indicating in vivo platelet activation. Concentrations of 5-HT in PF of colic horses with compromised bowel were significantly lower than the corresponding plasma concentrations (P = 0.005). POTENTIAL RELEVANCE: In horses with compromised bowel, significant amounts of 5-HT can be released into the systemic circulation, through massive release of platelet-stored 5-HT. 5-HT is a very potent proinflammatory, vasoconstrictive and immunomodulatory agent. In view of the rapid and prolonged tachyphylaxia, shown for the jejunal 5-HT1A-like receptors, this increased systemic 5-HT release could play a role in the pathophysiology of ileus in horses.


Assuntos
Líquido Ascítico/química , Cólica/veterinária , Doenças dos Cavalos/metabolismo , Íleus/veterinária , Serotonina/metabolismo , Animais , Líquido Ascítico/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Cólica/sangue , Cólica/metabolismo , Cólica/cirurgia , Feminino , Doenças dos Cavalos/sangue , Doenças dos Cavalos/cirurgia , Cavalos , Íleus/sangue , Íleus/metabolismo , Íleus/cirurgia , Masculino , Ativação Plaquetária , Complicações Pós-Operatórias/veterinária , Serotonina/sangue
13.
Neurogastroenterol Motil ; 20(6): 700-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18248582

RESUMO

This study introduces a novel, simplified method for the evaluation of murine intestinal transit and contractility using fluorescence and video imaging. Intestinal transit was measured by evaluating the intestinal distribution of non-absorbable fluorescein-labelled dextran (70 kDa, FD70) along the gastrointestinal (GI) tract. After excision of the GI tract, two full-field images--one in normal illumination mode and another in fluorescent mode--were taken with a charge coupled device (CCD) camera and subsequently matched for calculation of fluorescence distribution along the GI tract. Immediately after, intestinal contractility was evaluated in different regions of the intact intestine by spatiotemporal motility mapping (i.e. video imaging). In control mice, the small intestine showed vigorous oscillatory contractions and FD70 was primarily distributed within the terminal ileum/caecum at 90 min postgavage. As validation step, the effect of intestinal manipulation (IM, surgical procedure) and two pharmacological agents--known to alter GI motility--was tested. At 24 h postoperatively, spontaneous contractile activity of the small intestine was nearly abolished in IM mice, leaving the small intestine distended and resulting in a significantly delayed intestinal transit. In accordance, spontaneous mechanical activity of circular muscle strips in standard organ baths was significantly reduced in IM mice compared to control mice. Administration of atropine (1-3 mg kg(-1), i.p.) suppressed spontaneous contractile activity along the entire intestinal tract and induced a dose-related delay in intestinal transit. In contrast, metoclopramide (3-10 mg kg(-1), i.p.) markedly increased contractile activity--however only in the upper GI tract--and accelerated intestinal transit in a dose-dependent manner.


Assuntos
Motilidade Gastrointestinal/fisiologia , Trânsito Gastrointestinal/fisiologia , Intestinos/fisiologia , Administração Oral , Animais , Dextranos , Diagnóstico por Imagem/métodos , Fluoresceínas , Fluorescência , Intestinos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Fatores de Tempo
14.
Equine Vet J ; 40(4): 313-20, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18267888

RESUMO

REASONS FOR PERFORMING STUDY: Prokinetic drugs used to treat gastrointestinal ileus in man have equivocal results in horses. In man, prokinetic drugs have 5-hydroxytryptamine4(5-HT4) receptors as their target, but little is known about the 5-HT-receptor subtypes in the equine small intestine. OBJECTIVE: Functional and immunohistochemical identification of the serotonin receptor subtype(s) responsible for the 5-HT induced contractile response in the equine circular jejunum. METHODS: Isometric organ-bath recordings were carried out to assess spontaneous and drug-evoked contractile activity of equine circular jejunum. Histological investigations by immunofluorescence analyses were performed to check for presence and localisation of this functionally identified 5-HT receptor subtype. RESULTS: Tonic contractions were induced by 5-HT in horse jejunal circular muscle. Tetrodotoxin, atropine and NG-nitro L-arginine did not modify this response. A set of 5-HT receptor subtype selective antagonists excluded interaction with 5-HT1B, 1D, 2A, 3, 4 and 7 receptors. The selective 5-HT1A receptor antagonists WAY 100635 and NAN 190 caused a clear rightward shift of the concentration-response curve to 5-HT. The contractile effect of 5-CT, that can interact with 5-HT1A, 1B, 1D, 5 and 7 receptors was also antagonised by WAY 100635, identifying the targeted 5-HT receptor as a 5-HT1A-like receptor. Immunohistology performed with rabbit polyclonal anti-5-HT1A receptor antibodies confirmed the presence of muscular 5-HT1A receptors in the muscularis mucosae, and both longitudinal and circular smooth muscle layers of the equine jejunum. CONCLUSIONS: Contractile responses in equine jejunal circular smooth muscle induced by 5-HT involves 5-HT1A-like receptors.


Assuntos
Doenças dos Cavalos/tratamento farmacológico , Íleus/veterinária , Contração Muscular/efeitos dos fármacos , Receptor 5-HT1A de Serotonina , Serotoninérgicos/farmacologia , Serotonina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Doenças dos Cavalos/patologia , Cavalos , Íleus/tratamento farmacológico , Íleus/patologia , Imuno-Histoquímica/veterinária , Jejuno , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/patologia , Receptor 5-HT1A de Serotonina/isolamento & purificação , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1A de Serotonina/fisiologia
15.
Neurogastroenterol Motil ; 20(2): 99-112, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18199093

RESUMO

5-Hydroxytryptamine(4) (5-HT(4)) receptors are an interesting target for the management of patients in need of gastrointestinal (GI) promotility treatment. They have proven therapeutic potential to treat patients with GI motility disorders. Lack of selectivity for the 5-HT(4) receptor has limited the clinical success of the agonists used until now. For instance, next to their affinity for 5-HT(4) receptors, both cisapride and tegaserod have appreciable affinity for other receptors, channels or transporters [e.g. cisapride: human ether-a-go-go-related gene (hERG) is K(+) channel and tegaserod: 5-HT(1) and 5-HT(2) receptors]. Adverse cardiovascular events observed with these compounds are not 5-HT(4) receptor-related. Recent efforts have led to the discovery of a series of selective 5-HT(4) receptor ligands, with prucalopride being the most advanced in clinical development. The selectivity of these new compounds clearly differentiates them from the older generation compounds by minimizing the potential of target-unrelated side effects. The availability of selective agonists enables the focus to shift to the exploration of 5-HT(4) receptor-related differences between agonists. Based on drug- and tissue-related properties (e.g. differences in receptor binding, receptor density, effectors, coupling efficiency), 5-HT(4) receptor agonists are able to express tissue selectivity, i.e. behave as a partial agonist in some and as a full agonist in other tissues. Furthermore, the concept of ligand-directed signalling offers great opportunities for future drug development by enlarging the scientific basis for the generation of agonist-specific effects in different cell types, tissues or organs. Selective 5-HT(4) receptor agonists might thus prove to be innovative drugs with an attractive safety profile for better treatment of patients suffering from hypomotility disorders.


Assuntos
Fármacos Gastrointestinais/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Agonistas do Receptor 5-HT4 de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Animais , Ensaios Clínicos como Assunto , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/fisiopatologia , Humanos
16.
Neuropharmacology ; 53(2): 257-71, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17612577

RESUMO

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration-response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K(+) channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y(1), P2Y(6) and P2X(1) receptors on smooth muscle cells and P2Y(2), P2Y(12), P2X(2) and P2X(3) receptors in the myenteric plexus; almost a quarter of the P2Y(2)-immunopositive neurons co-expressed nNOS. The P2X-selective agonist alphabetameATP and the P2Y-selective agonist ADPbetaS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPbetaS was antagonised by the P2Y(1)-selective antagonist MRS2179 pointing to interaction with the muscular P2Y(1)-receptors. The relaxant effect of alphabetameATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X(1)-selective antagonist NF279; this correlates with an interaction with neuronal P2X(3) and muscular P2X(1) receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y(2)-receptors inducing relaxation, at least partially, by NO release.


Assuntos
Colo/citologia , Músculo Liso/fisiologia , Antagonistas de Receptores Purinérgicos P1 , Receptores Purinérgicos P1/fisiologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Anestésicos Locais/farmacologia , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Técnicas In Vitro , Masculino , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Agonistas do Receptor Purinérgico P1 , Ratos , Ratos Wistar , Receptores Purinérgicos P1/classificação , Tetrodotoxina/farmacologia , Tionucleotídeos/farmacologia
17.
Neurogastroenterol Motil ; 17(3): 366-75, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15916624

RESUMO

5-Hydroxytryptamine 4 (5-HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5-HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5-HT4 receptor-mediated stimulation of human colon CM contractile activity. 5-HT4 receptor ligands were tested on electrical field stimulation (EFS)-induced contractions of human colonic muscle strips cut in the circular direction (called 'whole tissue' strips). Additionally, after incubation of tissues with [3H]-choline these compounds were tested on EFS-induced release of tritium in whole tissue strips and in 'isolated' CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS-induced contractions of whole tissue CM strips. Prucalopride (0.3 micromol L-1) evoked a heterogenous response on EFS-induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS-induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS-induced tritium and [3H]-acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5-HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5-HT4 receptors on cholinergic nerves within the CM of human colon.


Assuntos
Colo/inervação , Colo/fisiologia , Músculo Liso/inervação , Músculo Liso/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Receptores 5-HT4 de Serotonina/efeitos dos fármacos , Acetilcolina/metabolismo , Benzofuranos/farmacologia , Cromatografia Líquida de Alta Pressão , Colo/metabolismo , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Tetrodotoxina/farmacologia
18.
Neurogastroenterol Motil ; 17(2): 229-35, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15810175

RESUMO

The influence of non-selective nitric oxide synthase (NOS) inhibition on gastric emptying of a semi-solid meal was studied in conscious pigs. Antroduodenal motility and fundic compliance were also assessed to evaluate the mechanisms at the origin of potential alteration in gastric emptying pattern. N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg kg(-1) i.v.) delayed gastric emptying (half-emptying time of 128.98 +/- 16.86 min vs 73.74 +/- 7.73 min after saline, P < 0.05, n = 6) as a result of decreased proximal gastric emptying. No changes were observed for distal gastric emptying as a result of unchanged antral motility. Similarly, no changes were noted on duodenal motor patterns either in the fasted or in the fed state. L-NAME decreased fundic compliance in fasted state (49 +/- 11 mL mmHg(-1) vs 118 +/- 15 mL mmHg(-1) after saline, P < 0.05, n = 6). As this phenomenon is expected to increase emptying rate, the gastroparesis induced by NOS inhibition is thus likely to originate from distal resistive forces. It is concluded that NO positively modulates gastric emptying.


Assuntos
Esvaziamento Gástrico/fisiologia , Óxido Nítrico/metabolismo , Estômago/fisiologia , Animais , Pressão Sanguínea/fisiologia , Complacência (Medida de Distensibilidade) , Inibidores Enzimáticos/farmacologia , Feminino , Contração Muscular/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Suínos
19.
Vet Q ; 27(4): 157-72, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16402513

RESUMO

OBJECTIVE: Hypocalcaemia is a common finding in horses with enterocolitis and severe gastrointestinal disorders. The aims of this study were to investigate in colic horses 1)the parameters related to hypocalcaemia, 2)the influence of hypocalcaemia on outcome and 3)the possible beneficial effect of Ca2+ substitution. DESIGN: Randomized controlled trial. SETTING: Intensive care unit. PATIENTS: One hundred forty-four horses that were admitted with an acute abdomen during a 1.5 year period were enrolled and daily evaluated for clinical criteria and whole blood ionized Ca2+ levels. Colic horses with hypocalcaemia were randomly assigned to receive Ca2+. INTERVENTIONS: Analysis of heparinised whole blood samples. Horses that were assigned to be treated received 400 mEq Ca2+ diluted in 10L of Ringer's lactate solution every 24 h until low reference range limits were obtained or until death. MEASUREMENTS AND MAIN RESULTS: 88% of all colic patients showed blood ionized Ca2+ levels below the reference range at the time of admission. Multivariable analysis revealed that the presence of reflux, signs of endotoxaemia, increased Packed Cell Volume (PCV), alkalinization of pH and the interaction PCV/pH all predispose colic horses to low ionized Ca2+ levels at the time of admission. The Odds for developing ileus during hospitalization are +/- 11.94 times larger for horses in the "very low" calcaemia interval, in comparison with normocalcaemic horses. The Odds for fatal outcome are respectively +/- 9.82 and 8.33 times larger for horses in the "very low" and "low" calcaemia interval. Ca2+ substitution increased the probability of survival, provided that Ca2+ levels could be normalized. The lack of an upward calcaemia response, despite repetitive Ca2+ substitutions, can be guarded as a poor ominous sign. CONCLUSIONS: Hypocalcaemia in colic horses is of prognostic relevance both with regard to survival as to the probability of development of ileus during hospitalization. This study shows the importance of routine measurement of ionized calcium levels in colic horses. Moreover, correction of hypocalcaemia seems to improve clinical outcome.


Assuntos
Cálcio/sangue , Cálcio/uso terapêutico , Cólica/veterinária , Doenças dos Cavalos/sangue , Hipocalcemia/veterinária , Íleus/veterinária , Abdome Agudo/sangue , Abdome Agudo/tratamento farmacológico , Abdome Agudo/veterinária , Animais , Cólica/sangue , Cólica/complicações , Cólica/tratamento farmacológico , Feminino , Refluxo Gastroesofágico/sangue , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/veterinária , Hematócrito/veterinária , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/etiologia , Cavalos , Concentração de Íons de Hidrogênio , Hipocalcemia/sangue , Hipocalcemia/complicações , Hipocalcemia/tratamento farmacológico , Íleus/epidemiologia , Íleus/etiologia , Masculino , Análise Multivariada , Prognóstico , Análise de Sobrevida
20.
Curr Pharm Des ; 10(20): 2483-97, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15320758

RESUMO

Gastrointestinal (GI) smooth muscle cell activity is controlled by contractile cholinergic neurons and relaxant non-adrenergic non-cholinergic (NANC) neurons in the myenteric plexus between the circular and longitudinal muscle layer. Decreased or increased NANC relaxation might be involved in the pathophysiology of functional GI motility disorders. Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) are the primary inhibitory NANC neurotransmitters. As classic neurotransmitters, VIP is stored in vesicles in the nerve endings, while NO is synthetized on demand by the neuronal isoform of NO synthase (nNOS). The VIP/nNOS co-localization in myenteric neurons, reported for various regions of the GI tract in different species, suggests that VIP and NO are co-transmitters. At the presynaptic level, VIP and NO can induce each others release. Most clear-cut evidence for this mechanism was obtained in isolated myenteric ganglia where VIP induced NO release, and NO facilitated VIP release. At the postsynaptic level, many studies support that VIP and NO are parallel co-transmitters, acting via the adenylate cyclase/3'5' adenosine cyclic monophosphate (cAMP) and guanylate cyclase/3'5' cyclic guanosine monophosphate pathway respectively. Mainly based on results obtained in isolated GI smooth muscle cells, a serial postsynaptic VIP/NO interaction model was proposed, whereby VIP is the principle neurotransmitter, acting partially via a VPAC receptor and the adenylate cyclase/cAMP pathway but also by induction of muscular NO production. Recent results suggest that the capacity of VIP to release NO from isolated smooth muscle cells is related to the induction of inducible NOS (iNOS) in the cells during the isolation procedure. The relative contribution of NO and VIP to GI NANC relaxation differs upon tissue and nerve firing frequency, so that interference with either of them will lead to varying effects.


Assuntos
Trato Gastrointestinal/fisiologia , Relaxamento Muscular/fisiologia , Músculo Liso/fisiologia , Óxido Nítrico/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Interações Medicamentosas , Trato Gastrointestinal/citologia , Trato Gastrointestinal/inervação , Humanos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Vias Neurais/anatomia & histologia , Vias Neurais/química , Vias Neurais/fisiologia , Neurônios/química , Neurônios/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/efeitos dos fármacos , Receptores de Peptídeo Intestinal Vasoativo/fisiologia
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