RESUMO
BACKGROUND AND PURPOSE: ET-1 signalling modulates intestinal motility and inflammation, but the role of ET-1/ETB receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ETB signalling regulates neural-motor pathways of intestinal motility and inflammation. EXPERIMENTAL APPROACH: We studied ETB signalling using: ETB drugs (ET-1, SaTX, BQ788), activity-dependent stimulation of neurons (high K+ -depolarization, EFS), gliotoxins, Tg (Ednrb-EGFP)EP59Gsat/Mmucd mice, cell-specific mRNA in Sox10CreERT2 ;Rpl22-HAflx or ChATCre ;Rpl22-HAflx mice, Sox10CreERT2 ::GCaMP5g-tdT, Wnt1Cre2 ::GCaMP5g-tdT mice, muscle tension recordings, fluid-induced peristalsis, ET-1 expression, qPCR, western blots, 3-D LSM-immunofluorescence co-labelling studies in LMMP-CM and a postoperative ileus (POI) model of intestinal inflammation. KEY RESULTS: In the muscularis externa ETB receptor is expressed exclusively in glia. ET-1 is expressed in RiboTag (ChAT)-neurons, isolated ganglia and intra-ganglionic varicose-nerve fibres co-labelled with peripherin or SP. ET-1 release provides activity-dependent glial ETB receptor modulation of Ca2+ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca2+ responses and excitatory cholinergic contractions, sensitive to L-NAME. Gliotoxins disrupt SaTX-induced glial-Ca2+ waves and prevent BQ788 amplification of contractions. The ETB receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ETB up-regulation, SaTX-hypersensitivity and glial amplification of ETB signalling. In vivo BQ788 (i.p., 1 mg·kg-1 ) attenuates intestinal inflammation in POI. CONCLUSION AND IMPLICATIONS: Enteric glial ET-1/ETB signalling provides dual modulation of neural-motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ETB receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI.
Assuntos
Gliotoxina , Íleus , Camundongos , Animais , Gliotoxina/metabolismo , Neuroglia , Neurônios/metabolismo , Íleus/tratamento farmacológico , Íleus/etiologia , Íleus/metabolismo , Motilidade Gastrointestinal , Inflamação/metabolismo , Colinérgicos/metabolismoRESUMO
Enteric glial cells (EGC) modulate motility, maintain gut homeostasis, and contribute to neuroinflammation in intestinal diseases and motility disorders. Damage induces a reactive glial phenotype known as "gliosis", but the molecular identity of the inducing mechanism and triggers of "enteric gliosis" are poorly understood. We tested the hypothesis that surgical trauma during intestinal surgery triggers ATP release that drives enteric gliosis and inflammation leading to impaired motility in postoperative ileus (POI). ATP activation of a p38-dependent MAPK pathway triggers cytokine release and a gliosis phenotype in murine (and human) EGCs. Receptor antagonism and genetic depletion studies revealed P2X2 as the relevant ATP receptor and pharmacological screenings identified ambroxol as a novel P2X2 antagonist. Ambroxol prevented ATP-induced enteric gliosis, inflammation, and protected against dysmotility, while abrogating enteric gliosis in human intestine exposed to surgical trauma. We identified a novel pathogenic P2X2-dependent pathway of ATP-induced enteric gliosis, inflammation and dysmotility in humans and mice. Interventions that block enteric glial P2X2 receptors during trauma may represent a novel therapy in treating POI and immune-driven intestinal motility disorders.
Assuntos
Gliose , Neuroglia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Animais , Citocinas , Inflamação , Intestino Delgado/fisiopatologia , CamundongosRESUMO
Early-life adversity contributes to the development of functional bowel disorders later in life through unresolved mechanisms. Here, we tested the hypothesis that early-life adversity alters anatomical and functional interactions between mast cells and enteric glia. The effects of early-life stress were studied using the neonatal maternal separation (NMS) stress mouse model. Anatomical relationships between mast cells and enteric glia were assessed using immunohistochemistry and mast cell reporter mice (Mcpt5Cre;GCaMP5g-tdT). Immunohistochemistry was used to assess the expression of histamine, histamine 1 (H1) receptors, and glial fibrillary acidic protein. Functional responses of glia to mast cell mediators were assessed in calcium imaging experiments using Sox10CreERT2;GCaMP5g-tdT mice and cultured human enteric glial cells. NMS increases mast cell numbers at the level of the myenteric plexus and their proximity to myenteric ganglia. Myenteric glia respond to mediators released by activated mast cells that are blocked by H1 receptor antagonists in mice and humans and by blocking neuronal activity with tetrodotoxin in mouse tissue. Histamine replicates the effects of mast cell supernatants on enteric glia, and NMS increases histamine production by mast cells. NMS reduces glial responses to mast cell mediators in mouse tissue, while potentiating responses in cultured human enteric glia. NMS increases myenteric glial fibrillary acidic protein expression and reduces glial process length but does not cause neurodegeneration. Histamine receptor expression is not altered by NMS and is localized to neurons in mice, but glia in humans. Early-life stress increases the potential for interactions between enteric glia and mast cells, and histamine is a potential mediator of mast cell-glial interactions through H1 receptors. We propose that glial-mast cell signaling is a mechanism that contributes to enteric neuroplasticity driven by early-life adversity.NEW & NOTEWORTHY Early-life adversity places an individual at risk for developing functional gastrointestinal disorders later in life through unknown mechanisms. Here, we show that interactions between mast cells and glia are disrupted by early-life stress in mice and that histamine is a potential mediator of mast cell-glial interactions.
Assuntos
Histamina/fisiologia , Acontecimentos que Mudam a Vida , Mastócitos/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Contagem de Células , Células Cultivadas , Quimases/genética , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Privação Materna , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Gravidez , Receptores Histamínicos H1/metabolismo , Estresse Psicológico/fisiopatologiaRESUMO
Mechanisms resulting in abdominal pain include altered neuro-immune interactions in the gastrointestinal tract, but the signaling processes that link immune activation with visceral hypersensitivity are unresolved. We hypothesized that enteric glia link the neural and immune systems of the gut and that communication between enteric glia and immune cells modulates the development of visceral hypersensitivity. To this end, we manipulated a major mechanism of glial intercellular communication that requires connexin-43 and assessed the effects on acute and chronic inflammation, visceral hypersensitivity, and immune responses. Deleting connexin-43 in glia protected against the development of visceral hypersensitivity following chronic colitis. Mechanistically, the protective effects of glial manipulation were mediated by disrupting the glial-mediated activation of macrophages through the macrophage colony-stimulating factor. Collectively, our data identified enteric glia as a critical link between gastrointestinal neural and immune systems that could be harnessed by therapies to ameliorate abdominal pain.
Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , Neuroglia/metabolismo , Animais , Humanos , Camundongos , FenótipoRESUMO
BACKGROUND: Clopidogrel is an irreversible antagonist of P2Y12 receptors (P2Y12Rs) used as an antiplatelet drug to reduce risk of thrombosis. P2Y12Rs are expressed in gastrointestinal (GI) tract where they might regulate GI function. AIM: To evaluate if blockade of P2Y12Rs by clopidogrel is associated with higher incidence of GI symptoms in patients with irritable bowel syndrome (IBS). METHODS: A retrospective analysis of our institutional database was conducted for a 13-year period. IBS patients were identified, and their demographics, GI symptoms and clopidogrel therapy were collected. Logistic regression models were used to characterize symptoms in clopidogrel versus no-clopidogrel IBS-groups, adjusting for Age and Sex differences. An additional study characterized the P2Y12R distribution in human gut. RESULTS: The search identified 7217 IBS patients (6761 no-clopidogrel/456 clopidogrel). There were a higher proportion of patients with GI symptoms on clopidogrel (68%) compared to controls (60%, p = 0.0011) that were Females (70 vs. 60%, p = 0.0003) not Males (61 vs. 60%; p = 0.8312). In Females, clopidogrel was associated with higher incidence of GI symptoms (Age adjusted; p < 0.0001) for pain, constipation, gastroparesis (p ≤ 0.0001) and psychogenic pain (p = 0.0006). Age or Sex (adjusted models) influenced one or more GI symptoms (i.e., pain, p < 0.0001; constipation, p < 0.0001/p = 0.008; diarrhea, flatulence, p = 0.01). P2Y12R immunoreactivity was abundant in human ENS; glial-to-neuron ratio of P2Y12Rs expressed in Females â« Males. CONCLUSIONS: Irreversible blockade of P2Y12R by clopidogrel is associated with higher incidence of GI symptoms in Female IBS patients, although Age or Sex alone contributes to symptomatology. Prospective studies can determine clinical implications of P2Y12Rs in IBS.
Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Intestinos/inervação , Síndrome do Intestino Irritável/epidemiologia , Inibidores da Agregação Plaquetária/efeitos adversos , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Ticlopidina/análogos & derivados , Dor Abdominal/induzido quimicamente , Dor Abdominal/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Clopidogrel , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/epidemiologia , Bases de Dados Factuais , Diarreia/induzido quimicamente , Diarreia/epidemiologia , Registros Eletrônicos de Saúde , Sistema Nervoso Entérico/química , Sistema Nervoso Entérico/fisiopatologia , Feminino , Flatulência/induzido quimicamente , Flatulência/epidemiologia , Gastroparesia/induzido quimicamente , Gastroparesia/epidemiologia , Humanos , Incidência , Síndrome do Intestino Irritável/diagnóstico , Síndrome do Intestino Irritável/fisiopatologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Receptores Purinérgicos P2Y12/análise , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Ticlopidina/efeitos adversos , Fatores de Tempo , Adulto JovemRESUMO
Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca2+-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca2+imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca2+oscillations in BON. UTP evoked a biphasic concentration-dependent Ca2+response. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,ß-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca2+responses were blocked with inhibitors of PLC, IP3R, SERCA Ca2+pump, La3+sensitive Ca2+channels or chelation of intracellular free Ca2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca2+pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca2+currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K+ channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca2+buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y4/P2Y6R. Zero-Ca2+buffer augmented Ca2+responses and 5-HT release. Conclusion: UTP activates a predominant P2Y4R pathway to trigger Ca2+oscillations via internal Ca2+mobilization through a PLC/IP3/IP3R/SERCA Ca2+signaling pathway to stimulate 5-HT release; Ca2+influx is inhibitory. UTP-induced Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca2+oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release.
RESUMO
BACKGROUND: Clinical observations or animal studies implicate enteric glial cells in motility disorders, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal (GI) infections, postoperative ileus, and slow transit constipation. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the "reactive human enteric glial cell (rhEGC) phenotype" induced by inflammation, and probe its functional relevance. METHODS: Human enteric glial cells in culture from 15 GI-surgical specimens were used to study gene expression, Ca, and purinergic signaling by Ca/fluo-4 imaging and mechanosensitivity. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport protein, channel, antioxidant, and other pathways. A 24-hour treatment with lipopolysaccharide (200 µg/mL) and interferon-γ (10 µg/mL) was used to induce inflammation and study molecular signaling, flow-dependent Ca responses from 3 mL/min to 10 mL/min, adenosine triphosphate (ATP) release, and ATP responses. RESULTS: Treatment induced a "rhEGC phenotype" and caused up-regulation in messenger RNA transcripts of 58% of 107 genes analyzed. Regulated genes included inflammatory genes (54%/IP10; IFN-γ; CxCl2; CCL3; CCL2; C3; s100B; IL-1ß; IL-2R; TNF-α; IL-4; IL-6; IL-8; IL-10; IL-12A; IL-17A; IL-22; and IL-33), purine-genes (52%/AdoR2A; AdoR2B; P2RY1; P2RY2; P2RY6; P2RX3; P2RX7; AMPD3; ENTPD2; ENTPD3; and NADSYN1), channels (40%/Panx1; CHRNA7; TRPV1; and TRPA1), vesicular transporters (SYT1, SYT2, SNAP25, and SYP), transcription factors (relA/relB, SOCS3, STAT3, GATA_3, and FOXP3), growth factors (IGFBP5 and GMCSF), antioxidant genes (SOD2 and HMOX1), and enzymes (NOS2; TPH2; and CASP3) (P < 0.0001). Treatment disrupted Ca signaling, ATP, and mechanical/flow-dependent Ca responses in human enteric glial cells. ATP release increased 5-fold and s100B decreased 33%. CONCLUSIONS: The "rhEGC phenotype" is identified by a complex cascade of pro-inflammatory pathways leading to alterations of important molecular and functional signaling pathways (Ca, purinergic, and mechanosensory) that could disrupt GI motility. Inflammation induced a "purinergic switch" from ATP to adenosine diphosphate/adenosine/uridine triphosphate signaling. Findings have implications for GI infection, inflammatory bowel disease, postoperative ileus, motility, and GI disorders.
Assuntos
Cálcio/metabolismo , Gastroenteropatias , Expressão Gênica , Inflamação , Neuroglia/metabolismo , Receptores Purinérgicos/genética , Transdução de Sinais/genética , Trifosfato de Adenosina/metabolismo , Canais de Cálcio/genética , Proteínas de Transporte/genética , Caspase 3/genética , Células Cultivadas , Colo Sigmoide/citologia , Citocinas/genética , Citocinas/metabolismo , Sistema Nervoso Entérico/citologia , Gastroenteropatias/genética , Gastroenteropatias/metabolismo , Motilidade Gastrointestinal , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Heme Oxigenase-1/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferon gama/farmacologia , Jejuno/citologia , Lipopolissacarídeos/farmacologia , Mecanotransdução Celular/genética , Óxido Nítrico Sintase Tipo II/genética , Fenótipo , Receptores Purinérgicos/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Superóxido Dismutase/genética , Fatores de Transcrição/genética , Triptofano Hidroxilase/genética , Regulação para Cima/efeitos dos fármacos , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: Alterations in 5-hydroxytryptamine (HT) signaling in inflamed gut may contribute to pathogenesis of inflammatory bowel diseases. Adenosine 5'-triphosphate (ATP) regulates mucosal-mechanosensory reflexes and ATP receptors are sensitive to mucosal inflammation. Yet, it remains unknown whether ATP can modulate 5-HT signaling in enterochromaffin cells (EC). We tested the novel purinergic hypothesis that ATP is a critical autocrine regulator of EC mechanosensitivity and whether EC expression of ATP-gated P2X3-ion channels is altered in inflammatory bowel diseases. METHODS: Laser confocal (fluo-4) Ca imaging was performed in 1947 BON cells. Chemical stimulation or mechanical stimulation (MS) was used to study 5-HT or ATP release in human BON or surgical mucosal specimens, and purine receptors by reverse transcription-polymerase chain reaction, Western Blot, or P2X3-immunoreactivity in BON or 5-HT human EC (hEC) in 11 control and 10 severely inflamed ulcerative colitis (UC) cases. RESULTS: ATP or MS triggered Ca-transients or 5-HT release in BON. ATP or adenosine diphosphate increased 5-HT release 5-fold. MS caused ATP release, detected after 5'ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5'-monophosphate-evoked (P2Y1,12) or mechanically-evoked responses were blocked or augmented by a P2Y1,12 antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y12 antagonist, 2MeSAMP, augmented responses. A P2X1,3 agonist, α,ß-MeATP, triggered Ca responses, whereas a P2X1,2/3,3 antagonist, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5'ATP- labeling. In hEC, α,ß-MeATP stimulated 5-HT release. In UC, P2X3-immunoreactivity decreased from 15% to 0.2% of 5-HThECs. Human mucosa and BON expressed P2X1, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12R-messenger RNA transcripts. CONCLUSIONS: ATP is a critical determinant of mechanosensation and 5-HT release via autocrine activation of slow P2Y1-phospholipase C/inositol-1,4,5-triphosphate-Ca or inhibitory P2Y12-purinergic pathways, and fast ATP-gated P2X3-channels. UC downregulation of P2X3-channels (or A2B) is postulated to mediate abnormal 5-HT signaling.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Autócrina , Colite Ulcerativa/metabolismo , Células Enterocromafins/metabolismo , Mucosa Intestinal/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Receptores Purinérgicos P2X3/metabolismo , Serotonina/metabolismo , Células Cultivadas , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/cirurgia , Células Enterocromafins/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Mucosa Intestinal/efeitos dos fármacos , Receptores Purinérgicos P2X3/químicaRESUMO
BACKGROUND: Pharmacological studies suggest that adenosine A3AR influences motility and colitis. Functional A3â»/â»AR knockout mice were used to prove whether A3AR activation is involved in modulating either motility or colitis. METHODS: A3AR was probed by polymerase chain reaction (PCR) genotyping, Western blot, and immunochemistry. Motility was assessed in vivo by artificial bead-expulsion, stool-frequency, and FITC-dextran transit. Colitis was induced with dextran sodium sulfate (DSS) in A3â»/â»AR or wildtype (WT) age- and sex-matched controls. Progression of colitis was evaluated by histopathology, changes in myeloperoxidase (MPO), colon length, CD4(+) -cells, weight-loss, diarrhea, and the guaiac test. RESULTS: Goat anti-hu-A3 antiserum identified a 66 kDa immunogenic band in colon. A3AR-immunoreactivity is expressed in SYN(+) -nerve varicosities, s-100(+) -glia, and crypt cells, but not 5-HT(+) (EC), CD4(+) (T), tryptase(+) (MC), or muscle cells. A3AR immunoreactivity in myenteric ganglia of distal colon >> proximal colon by a ratio of 2:1. Intestinal transit and bead expulsion were accelerated in A3â»/â»AR mice compared to WT; stool retention was lower by 40%-60% and stool frequency by 67%. DSS downregulated A3AR in epithelia. DSS histopathology scores indicated less mucosal damage in AA3â»/â»AR mice than WT. A3â»/â»AR phenotype protected against DSS-induced weight loss, neutrophil (MPO), or CD4(+) -T cell infiltration, colon shortening, change in splenic weight, diarrhea, or occult-fecal blood. CONCLUSIONS: Functional disruption of A3AR in A3â»/â»AR mice alters intestinal motility. We postulate that ongoing release of adenosine and activation of presynaptic-inhibitory A3AR can slow down transit and inhibit the defecation reflex. A3AR may be involved in gliotransmission. In separate studies, A3â»/â»AR protects against DSS colitis, consistent with a novel hypothesis that A3AR activation contributes to development of colitis.
Assuntos
Colite/fisiopatologia , Colo/fisiopatologia , Trânsito Gastrointestinal/fisiologia , Receptor A3 de Adenosina/fisiologia , Animais , Contagem de Linfócito CD4 , Colite/induzido quimicamente , Colite/patologia , Colo/inervação , Colo/patologia , Defecação/fisiologia , Sulfato de Dextrana , Diarreia/fisiopatologia , Feminino , Genótipo , Camundongos , Camundongos Knockout , Plexo Mientérico/metabolismo , Sangue Oculto , Tamanho do Órgão , Peroxidase/metabolismo , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Baço/patologia , Fatores de Tempo , Redução de PesoRESUMO
We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that eADO acts at low-affinity A3 receptors in addition to high-affinity A1 receptors to suppress coordinated responses triggered by immune-histamine H2 receptor activation. The short interplexus circuit activated by histamine involves adenosine, acetylcholine, substance P, and serotonin. We postulate that A3 receptor modulation may occur in gut inflammatory diseases or allergic responses involving mast cell and histamine release.
Assuntos
Adenosina/metabolismo , Colo/metabolismo , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal , Histamina/metabolismo , Músculo Liso/metabolismo , Inibição Neural , Intestino Neurogênico/metabolismo , Receptor A3 de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Cloretos/metabolismo , Cimetidina/farmacologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/inervação , Di-Hidropiridinas/farmacologia , Dimaprit/farmacologia , Relação Dose-Resposta a Droga , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/fisiopatologia , Motilidade Gastrointestinal/efeitos dos fármacos , Cobaias , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Técnicas In Vitro , Secreções Intestinais/metabolismo , Masculino , Mecamilamina/farmacologia , Contração Muscular , Relaxamento Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/imunologia , Músculo Liso/inervação , Inibição Neural/efeitos dos fármacos , Intestino Neurogênico/imunologia , Intestino Neurogênico/fisiopatologia , Antagonistas dos Receptores de Neurocinina-1 , Antagonistas Nicotínicos/farmacologia , Piperidinas/farmacologia , Propano/análogos & derivados , Propano/farmacologia , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/efeitos dos fármacos , Receptores Histamínicos H2/efeitos dos fármacos , Receptores Histamínicos H2/metabolismo , Receptores da Neurocinina-1/metabolismo , Reflexo , Teofilina/análogos & derivados , Teofilina/farmacologia , Xantinas/farmacologiaRESUMO
BACKGROUND: Expression of purine genes is modulated by inflammation or experimental colitis and altered expression leads to disrupted gut function. We studied purine gene dysregulation profiles in inflammatory bowel disease (IBD) and determined whether they can distinguish between Crohn's disease (CD) and ulcerative colitis (UC) using Pathway Analysis and a new Comparative Analysis of Gene Expression and Selection (CAGES) method. METHODS: Raw datasets for 22 purine genes and 36 probe-sets from National Center for Biotechnology Information (NCBI) GEO (Gene Expression Omnibus) (http://www.ncbi.nlm.nih.gov/projects/geo/) were analyzed by National Cancer Institute (NCI) Biological Resources Branch (BRB) array tools for random-variance of multiple/36 t-tests in colonic mucosal biopsies or peripheral blood mononuclear cells (PBMCs) of CD, UC or control subjects. Dysregulation occurs in 59% of purine genes in IBD including ADORA3, CD73, ADORA2A, ADORA2B, ADAR, AMPD2, AMPD3, DPP4, P2RY5, P2RY6, P2RY13, P2RY14, and P2RX5. RESULTS: In CD biopsies, expression of ADORA3, AMPD3, P2RY13, and P2RY5 were negatively correlated with acute inflammatory score, Crohn's Disease Activity Index (CDAI) or disease chronicity; P2RY14 was positively correlated in UC. In mucosal biopsies or PBMCs, CD and UC were distinguished by unique patterns of dysregulation (up- or downregulation) in purine genes. Purine gene dysregulation differs between PBMCs and biopsies and possibly between sexes for each disease. Ingenuity Pathway Analysis (IPA) revealed significant associations between alterations in the expression of CD73 (upregulation) or ADORA3 (downregulation) and inflammatory or purine genes (Assuntos
Colite Ulcerativa/genética
, Colite Ulcerativa/metabolismo
, Doença de Crohn/genética
, Doença de Crohn/metabolismo
, Perfilação da Expressão Gênica
, Purinas/metabolismo
, Transdução de Sinais/genética
, Biópsia
, Doença Crônica
, Colite Ulcerativa/patologia
, Biologia Computacional
, Doença de Crohn/patologia
, Bases de Dados Genéticas
, Feminino
, Humanos
, Masculino
, Análise de Sequência com Séries de Oligonucleotídeos
, Receptor A3 de Adenosina/genética
, Receptor A3 de Adenosina/metabolismo
, Receptores A2 de Adenosina/genética
, Receptores A2 de Adenosina/metabolismo
, Receptores Purinérgicos P2/genética
, Receptores Purinérgicos P2/metabolismo
, Índice de Gravidade de Doença
RESUMO
Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Colite/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Colite/induzido quimicamente , Colite/enzimologia , Colite/genética , Modelos Animais de Doenças , Radicais Livres/metabolismo , Glutationa Peroxidase/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Ácido TrinitrobenzenossulfônicoRESUMO
BACKGROUND & AIMS: The aim was to determine whether adenosine receptors modulate cAMP, intracellular free calcium ([Ca(2+)](i)), and 5-hydroxytryptamine (5-HT) release in human carcinoid BON cells. METHODS: Adenosine receptor (R) mRNA, proteins, and function were identified by Western blots, immunofluorescent labeling, Fluo-4/AM [Ca(2+)](i) imaging, and pharmacologic/physiologic techniques. RESULTS: A1, A2, and A3Rs were present in BON cells and carcinoid tumors. Baseline 5-HT levels increased with adenosine deaminase, activation of A2Rs, and inhibition of A3Rs, whereas A3R activation decreased 5-HT. A2R antagonists or blockade of adenosine reuptake that elevates extracellular adenosine reduced mechanically evoked 5-HT release. In single BON cells, touch elevated [Ca(2+)](i) responses were augmented by adenosine deaminase, A1, and A3R antagonists. CONCLUSIONS: Tonic or mechanically evoked release of endogenous adenosine is a critical determinant of differential activation of adenosine receptors and may have important implications for gut mechanosensory reflexes.