RESUMO
BACKGROUND: Clostridium difficile (Cdf) releases toxins (TcdA and TcdB) that damage the intestinal epithelial barrier. Ecto-5'-nucleotidase (CD73) is expressed on intestinal epithelial cells, and it is hypothesized to protect against toxin-induced epithelial damage through the cleavage of 5'-AMP to adenosine (Ado) and subsequent activation of adenosine receptors (AdoRs). Herein, we sought to assess the potential protective effects of CD73 and AdoR signaling on the injurious effects of Cdf toxins. METHODS: Barrier function was assessed with T84 colonocytes. Transepithelial electrical resistance (TEER), paracellular fluorescein isothiocyanate (FITC)-dextran flux, and tight junction protein (ZO-1) integrity were monitored. Intrarectal installation of Cdf toxin was used to assess epithelial damage in vivo. KEY RESULTS: TcdA/B caused reduced TEER and increased paracellular flux in vitro. Concurrent treatment with 5'-AMP attenuated these responses to Cdf toxin; an effect that was blocked with ZM241385 (AdoRA2 antagonist). APCP, a CD73 inhibitor, also suppressed the protective effects of 5'-AMP on paracellular flux. 5'-AMP reduced toxin-induced disruption of ZO-1, an effect that was abolished by APCP and ZM241385. Inhibition of CD73 with APCP during Cdf toxin exposure led to increased intestinal barrier permeability and epithelial damage in vivo. Intrarectal instillation of 5'-AMP had no effect on toxin-induced intestinal injury. CONCLUSIONS & INFERENCES: Our data suggest that CD73 has a protective role against TcdA/B-induced damage. 5'-AMP treatment attenuated the damaging effects of Cdf toxin in vitro, and inhibitors of CD73 (APCP) and AdoRs (ZM241385) revealed that the cleavage of 5'-AMP to Ado was necessary for the protective effects. Inhibition of CD73 in vivo increases colonic tissue damage and epithelial permeability during Cdf toxin exposure.
Assuntos
5'-Nucleotidase/metabolismo , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Enterotoxinas/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Receptores Purinérgicos P1/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Permeabilidade , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
BACKGROUND: Salvinorin A (SA) is the principal active ingredient of Salvia divinorum, with an established inhibitory action on gastrointestinal (GI) transit and colonic ion transport in mice. Under normal conditions, the effects of SA are mediated by kappa opioid (KOR) and cannabinoid (CB1 and CB2) receptors. However, the role of SA in pathophysiological conditions remains unresolved. The aim of this study was to characterize the in vitro and in vivo effects of SA on mouse ileum after endotoxin challenge. METHODS: Changes in GI motility were studied in vitro, using smooth muscle preparations from the mouse ileum. In vivo, the fecal pellet output and small intestinal fluid content were measured. Neurogenic ion transport and intestinal permeability were examined using Ussing chambers. In addition, Western blot analysis of mucosa was performed and plasma nitrite/nitrate levels were determined. KEY RESULTS: Salvinorin A inhibited endotoxin-induced ileal hypercontractility via KOR, CB1, and CB2 receptors. Neurogenic ion transport, which was significantly reduced after endotoxin challenge, was normalized by SA through a nitric oxide synthase (NOS)-dependent mechanism. Western blot analysis and plasma nitrite/nitrate level quantitation confirmed the involvement of NOS in the regulatory action of SA. CONCLUSIONS & INFERENCES: This is the first report showing differential effects of SA on motor and secretory activity in mouse GI during endotoxemia. The outcomes of our study imply possible novel applications of SA and its analogs in the treatment of GI disorders.
Assuntos
Diterpenos Clerodânicos/farmacologia , Endotoxinas/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Íleo/fisiologia , Transporte de Íons/efeitos dos fármacos , Animais , Defecação/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismoRESUMO
BACKGROUND: The gut plays a significant role in the development of obesity, notably through peptide signaling to the brain. However, few studies have investigated intestinal function per se in a rodent model of diet-induced obesity (DIO). Our aim was to investigate intestinal secretomotor function and glucose transport in DIO and diet-resistant (DR) rat jejunum. METHODS: Male outbred Sprague-Dawley rats were maintained on a medium high fat diet for 9-10 weeks and split into DIO and DR groups based on weight gain. Mucosal-submucosal preparations of the proximal jejunum were mounted in Ussing chambers and voltage-clamped at 0 mV. Glucose (10 mmol L(-1)), 2-deoxy-D-glucose (10 mmol L(-1)), and leptin (10 nmol L(-1)) were added to the luminal side of the tissue and veratridine (30 micromol L(-1)), bethanechol (100 micromol L(-1)), and forskolin (10 micromol L(-1)) were added to the basolateral side of the tissue. KEY RESULTS: Secretomotor responses were significantly decreased in DIO jejunum compared to DR tissues. Glucose-stimulated increases in I(sc) in DR animals, that were sensitive to leptin inhibition, were significantly reduced in DIO rats. Decreased sodium glucose transporter-1 mediated glucose transport was accompanied by a concomitant increase in the expression of jejunal glucose transporter-2. CONCLUSIONS & INFERENCES: These data suggest that submucosal nerve function is compromised in DIO rats and electrogenic glucose transport is significantly decreased. The latter may represent an adaptive response to limit nutrient absorption in the jejunum from DIO rats. However, the loss of secretomotor control may lead to an altered host defense with a resultant change in intestinal flora contributing to the maintenance of obesity.
Assuntos
Dieta , Alimentos , Absorção Intestinal/fisiologia , Intestinos/fisiologia , Obesidade/fisiopatologia , Animais , Western Blotting , Motilidade Gastrointestinal/fisiologia , Glucose/metabolismo , Glucose/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Jejuno/fisiologia , Leptina/sangue , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists. METHODS: Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin. KEY RESULTS: In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. CONCLUSIONS & INFERENCES: Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.
Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Colo/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Fezes/química , Trânsito Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Imuno-Histoquímica , Íons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/efeitos dos fármacos , Permeabilidade , Piperidinas/farmacologia , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/genética , Rimonabanto , Água/análiseRESUMO
The life expectancy for cystic fibrosis (CF) patients has increased dramatically over the last 30 years. Although the overall cancer risk for CF patients does not appear to be increased there is a marked increased risk of gastrointestinal malignancies especially in the post lung transplant population. CF patients that do develop gastrointestinal malignancies do so at an earlier age and there is often a lag in the diagnosis and management of these individuals. We present a 39 year old male CF patient that underwent a colonoscopy for colon cancer screening and a large, near obstructing, villous adenoma of his ileum was found. The polyp was removed successfully via endoscopy without incident and there was no evidence of malignancy. An upper endoscopy revealed a long segment of Barrett's esophagus with no evidence of dysplasia. We present this case as well as a detailed review of the literature on cancer risk in CF and a discussion of the mechanisms that may be involved. We also present the risk of GI malignancies in non-CF patients as a guide on how to assess and manage the risk of GI malignancies in this ever-changing patient population.
Assuntos
Adenoma Viloso/complicações , Esôfago de Barrett/complicações , Fibrose Cística/complicações , Neoplasias do Íleo/complicações , Adenoma Viloso/patologia , Adulto , Esôfago de Barrett/patologia , Colonoscopia , Endoscopia Gastrointestinal , Predisposição Genética para Doença , Humanos , Neoplasias do Íleo/patologia , Masculino , Fatores de RiscoRESUMO
PURPOSE: Both enteric infection and exposure to ionizing radiation are associated with increased intestinal permeability. However, the combined effect of irradiation and enteric infection has not been described. We combined infection of mice with the enteric pathogen, Citrobacter rodentium, with exposure to ionizing radiation and assessed the impact on colonic epithelial ion transport, permeability and bacterial translocation. MATERIALS AND METHODS: Mice were infected with C. rodentium and then received whole-body exposure to 5 Gray gamma-radiation 7 days later. Three days post-irradiation, mice were euthanized and colons removed. Control groups included sham-infected mice that were irradiated and mice that were infected, but not irradiated. RESULTS: Macroscopic damage score and colonic wall thickness were increased by C. rodentium infection, but these parameters were not exacerbated by irradiation. Infection caused an increase in myeloperoxidase activity that was reduced by irradiation. Irradiation reduced the secretory response to electrical field stimulation, forskolin and carbachol; these changes were not altered by infection with C. rodentium. None of the treatments caused an increase in permeability to 51Cr-ethylenediaminetetraacetic acid (EDTA). However, combined infection and irradiation synergistically increased bacterial translocation to mesenteric lymph nodes, liver, spleen and blood. CONCLUSIONS: Although the combination of irradiation and infection did not exacerbate the individual effects of these challenges on ion secretion and mucosal permeability to 51Cr-EDTA, it dramatically increased susceptibility to bacterial translocation and bacteremia. These results have important implications for patients who develop an enteric infection during the course of abdominopelvic radiotherapy.
Assuntos
Translocação Bacteriana/efeitos da radiação , Citrobacter rodentium/fisiologia , Citrobacter rodentium/efeitos da radiação , Colite/microbiologia , Colo/microbiologia , Colo/efeitos da radiação , Infecções por Enterobacteriaceae/microbiologia , Animais , Suscetibilidade a Doenças/microbiologia , Relação Dose-Resposta à Radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doses de Radiação , Radiação IonizanteRESUMO
Colitis is associated with alterations in electrolyte and water transport. These changes give rise to some of the symptoms experienced by patients with colitis. Alterations in fluid flux may also contribute to increased susceptibility to mucosal injury. Recently, endogenous water channel proteins (aquaporins; AQPs), have been identified in colonic tissue. The expression of AQP4, AQP7 and AQP8 was examined, via reverse transcription/polymerase chain reaction, Western blotting and immunohistochemistry, in a murine model of colitis and in patients with inflammatory bowel disease or infectious colitis. Colitis was induced in C57BL/6 mice by the addition of 2.5% dextran sodium sulphate (DSS) to their drinking water. AQP expression in these mice was assessed following 12 h to 7 days of DSS exposure and during the recovery phase from 1 to 15 days following cessation of DSS exposure. Colonic water transport was measured after 1 and 3 days of DSS and following 7 days of recovery. The expression of AQP4 and AQP8 mRNA was significantly decreased after 12-24 h of DSS exposure and remained depressed throughout the treatment period. Expression of AQP7 was more variable. Protein expression followed a similar pattern to that observed for AQP mRNA. Significant alteration in colonic fluid secretion was correlated with reduced expression of AQP isoforms. Significantly, patients with active ulcerative colonic, Crohn's colitis or infectious colitis had similar dramatic reductions in AQP expression that appeared to be correlated with disease activity. Thus, colonic injury in both mouse and man is associated with a downregulation in AQP expression.
Assuntos
Aquaporinas/metabolismo , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Regulação para Baixo , Animais , Aquaporinas/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Doença de Crohn/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The presence of neuronal reflexes within the intestine that modulate mucosal ion secretion and blood flow have been recognized for many years, but the organization of these reflexes was unclear. This review highlights important findings from recent in vitro guinea-pig studies which have shown that both intrinsic primary afferent neurones (IPANs) and extrinsic primary afferent neurones (EPANs) can respond to chemical and/or mechanical stimuli to activate pathways, the afferent and efferent elements of which are confined to the walls of the intestine. Enteric neuronal pathways involve both myenteric and submucosal plexus neurones whereas capsaicin-sensitive afferent nerves evoke secretion by stimulating submucosal secretomotor neurones and vasodilation by direct actions on the submucosal arterioles. In this review, the cellular mechanisms involved in these pathways are described and the implications of these findings are discussed.
Assuntos
Sistema Digestório/inervação , Neurônios Aferentes/fisiologia , Reflexo/fisiologia , Plexo Submucoso/citologia , Plexo Submucoso/fisiologia , Animais , Cobaias , Transmissão Sináptica/fisiologia , Vasodilatação/fisiologiaRESUMO
Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.
Assuntos
Inflamação/metabolismo , Intestinos/fisiologia , Glicoproteínas de Membrana/biossíntese , Plexo Mientérico/metabolismo , Animais , Western Blotting , Colite/induzido quimicamente , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Proteína Ligante Fas , Trânsito Gastrointestinal/fisiologia , Cobaias , Ileíte/induzido quimicamente , Ileíte/imunologia , Ileíte/metabolismo , Imuno-Histoquímica , Inflamação/imunologia , Intestinos/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Plexo Mientérico/imunologia , Neutrófilos/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
BACKGROUND: Hyporesponsiveness of the intestinal epithelium to secretagogues occurs in different models of intestinal injury, including radiation enteropathy, and in human disease. While this impairment of barrier function has been linked to increased inducible nitric oxide synthase (iNOS) activity, the cellular target of NO in this phenomenon is not known, although recent studies suggest that some isoforms of adenylate cyclase are inhibited by NO. AIMS: To determine adenylate cyclase isoform distribution in colonic epithelial cells and, in particular, the physiological significance of NO inhibitable adenylate cyclase isoforms 5 and 6 in radiation induced epithelial secretory dysfunction. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and immunohistochemistry were used to examine adenylate cyclase expression. The responsiveness of mouse colon to secretagogues 72 hours post-15 Gy gamma radiation or following in vitro exposure to NO donors was measured in Ussing chambers. Also, cAMP, cGMP, and ATP levels were measured. RESULTS: RT-PCR, immunocytochemistry, and immunohistochemistry showed that adenylate cyclase 5 was expressed in mouse colon, and isoforms 5 and 6 were expressed in human biopsies and intestinal epithelium. Pharmacological studies showed that these isoforms are functionally important in chloride secretion. NO mediated hyporesponsiveness to secretagogues is primarily a result of decreased adenylate cyclase activity, and not G(i) activation or decreased cellular ATP levels. CONCLUSIONS: NO inhibitable isoforms of adenylate cyclase are expressed in mouse and human secretory colonic epithelia, and appear to be the target of radiation induced NO to reduce the responsiveness to cAMP dependent secretagogues.
Assuntos
Adenilil Ciclases/metabolismo , Colo/efeitos da radiação , Mucosa Intestinal/efeitos da radiação , Isoenzimas/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/genética , Animais , Carbacol/farmacologia , Colinérgicos/farmacologia , Colforsina/farmacologia , Colo/enzimologia , Colo/metabolismo , Guanilato Ciclase/metabolismo , Imuno-Histoquímica/métodos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND AIMS: The colonic epithelium plays a key role in host defence. During colitis, epithelial function is impaired, leading to elevated bacterial translocation and exacerbation of inflammation. We previously documented perturbation of epithelial function, in terms of secretion and as a barrier to bacterial translocation, that persisted long after resolution of a bout of colitis in the rat. The mechanisms underlying the epithelial dysfunction are not completely understood. METHODS: Given the ability of prostaglandin (PG) D2 to suppress colonic epithelial secretion, we investigated the potential roles of this eicosanoid and of cyclooxygenase 2 (COX-2) in mediating post-colitis epithelial secretory and barrier dysfunction. RESULTS: Six weeks after induction of colitis with trinitrobenzene sulphonic acid, there was marked elevated synthesis of PGD2 and elevated COX-2 expression. Selective COX-2 inhibition abolished the increase in PGD2 synthesis. Colonic chloride secretory responses (in vitro) were significantly diminished relative to those in controls, a defect that was reversed by pre-exposure to a selective COX-2 inhibitor (celecoxib) but not to a selective COX-1 inhibitor (SC-560). The hyporesponsiveness was mimicked by pre-exposure of normal colonic tissue to PGD2, but not to its metabolite, 15-deoxy-Delta(12-14)PGJ2. The post-colitis rats exhibited a 10-fold increase in bacterial colonisation of the colon, and >3-fold increase in bacterial translocation. Twice daily treatment for one week with a selective COX-2 inhibitor (rofecoxib) did not affect bacterial colonisation but abolished the increase in bacterial translocation. CONCLUSIONS: These studies demonstrate an important role for COX-2, possibly via generation of PGD2, in mediating the prolonged epithelial secretory and barrier dysfunction after a bout of colitis in the rat.
Assuntos
Colite/metabolismo , Colo/metabolismo , Isoenzimas/metabolismo , Prostaglandina D2/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Translocação Bacteriana/fisiologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Mucosa Intestinal/metabolismo , Isoenzimas/antagonistas & inibidores , Masculino , Proteínas de Membrana , Ratos , Ratos WistarRESUMO
Epithelial secretion may play an important role in reducing bacterial colonization and translocation in intestine. If so, secretory dysfunction could result in increased susceptibility to infection and inflammation. We investigated whether long-term colonic secretory dysfunction occurs after a bout of colitis and if this is accompanied by an increase in bacterial colonization and translocation. Rats were studied 6 wk after induction of colitis with trinitrobenzene sulfonic acid when inflammation had completely resolved, and epithelial permeability was normal. Intestinal loops were stimulated with either Clostridium difficile toxin A or a phosphodiesterase inhibitor. In vitro, colonic tissue from previously sensitized rats was exposed to antigen (ovalbumin). Secretory responses to all three stimuli were suppressed in rats that had previously had colitis. These rats exhibited increased (16-fold) numbers of colonic aerobic bacteria and increased (>3-fold) bacterial translocation, similar to results in rats studied after resolution of enteritis. Postcolitis bacterial translocation was prevented by daily treatment with an inhibitor of inducible nitric oxide synthase. This study demonstrates that intestinal inflammation results in prolonged impairment of colonic epithelial secretion, which may contribute to increases in bacterial load and bacterial translocation. Epithelial dysfunction of this type could underlie an increased propensity for further bouts of inflammation, a hallmark of diseases such as inflammatory bowel disease.
Assuntos
Translocação Bacteriana , Colite/microbiologia , Colite/fisiopatologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiopatologia , 1-Metil-3-Isobutilxantina/farmacologia , Doença Aguda , Animais , Bactérias Aeróbias/isolamento & purificação , Toxinas Bacterianas/farmacologia , Translocação Bacteriana/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Doença Crônica , Colite/induzido quimicamente , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/fisiopatologia , Enterotoxinas/farmacologia , Inibidores Enzimáticos/farmacologia , Etanol , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Masculino , Muco/efeitos dos fármacos , Muco/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Ratos Wistar , Remissão Espontânea , Ácido TrinitrobenzenossulfônicoRESUMO
The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl(-)-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2 and the Ca(2+)-ATPase inhibitor thapsigargin, but not by the L-type Ca(2+) channel blocker verapamil or DIDS, the nonselective inhibitor of Ca(2+)-dependent Cl(-) transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca(2+). RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl(-) channel-2 but not for the Ca(2+)-dependent Cl(-) channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl(-) secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca(2+)-dependent Cl(-) secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.
Assuntos
Cálcio/fisiologia , Cloretos/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Trombina/fisiologia , Linhagem Celular , AMP Cíclico/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Condutividade Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Transporte de Íons , Oligopeptídeos/farmacologia , RNA Mensageiro/biossíntese , Receptor PAR-1 , Receptores de Trombina/genética , Trombina/farmacologiaRESUMO
PURPOSE: The studies were designed to investigate the differences in the intestinal inflammatory response following abdominopelvic or total-body irradiation in a ferret model. MATERIALS AND METHODS: Ferrets were exposed either to total-body or to abdominopelvic gamma-radiation (5 Gy) and various parameters of inflammation studied in the jejunum, ileum and colon 2 and 7 days later. RESULTS: Abdominopelvic and, to a greater extent, total-body irradiation caused weight loss by 7 days. White blood cell counts were reduced in both groups, but more so following total-body irradiation. Myeloperoxidase activity was significantly increased in the ileum 2 days after abdominopelvic irradiation, but it was reduced after total-body irradiation. Total-body irradiation increased tissue prostaglandin E2 levels in all regions at 2 days and decreased jejunal leukotriene B4 levels in the jejunum at both time points. Ileal prostaglandin E2 levels were increased 2 days after abdominopelvic irradiation. Expression of inducible nitric oxide synthase was not altered by either irradiation protocol. CONCLUSIONS: The data show that there are regional differences in the intestinal response to irradiation, depending on whether it was delivered to the whole body or locally to the abdominopelvic region. In particular, the ileum exhibited an acute increase in myeloperoxidase activity following abdominopelvic but not total-body irradiation.
Assuntos
Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos da radiação , Lesões Experimentais por Radiação/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Colo/metabolismo , Colo/efeitos da radiação , Dinoprostona/metabolismo , Furões , Íleo/metabolismo , Íleo/efeitos da radiação , Inflamação/etiologia , Jejuno/metabolismo , Jejuno/efeitos da radiação , Contagem de Leucócitos , Leucotrieno B4/metabolismo , Masculino , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Peroxidase/metabolismo , Lesões Experimentais por Radiação/complicaçõesRESUMO
The receptor responsible for CGRP-induced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentration-dependent increase in short circuit current (I(sc), EC(50) 21 nM), which was abolished in chloride-free buffer but was not blocked by CGRP(8-37) or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in I(sc). The responses to the linear CGRP(2) receptor agonists [Cys(Et)(2,7)] hCGRPalpha and [Cys(Acm)(2,7)] hCGRPalpha were considerably smaller than the response to CGRP. These responses were abolished in chloride-free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP(2) agonists. The response to [Cys(Et)(2,7)] hCGRPalpha was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (I(sc), EC(50) 15 nM) and [Cys(Et)(2,7)] hCGRPalpha as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte.
Assuntos
Colite/fisiopatologia , Colo/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/análogos & derivados , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Colite/induzido quimicamente , Colo/fisiopatologia , Condutividade Elétrica , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Tetrodotoxina/farmacologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Sangre de grado is an Amazonian herbal medicine used to facilitate the healing of gastric ulcers and to treat gastritis, diarrhea, skin lesions, and insect stings. This study was designed to evaluate the gastrointestinal applications. Gastric ulcers were induced in rats by brief serosal exposure of the fundus to acetic acid (80%). Sangre de grado was administered in drinking water at 1:1,000 and 1:10,000 dilutions from the postoperative period to day 7. Guinea pig ileum secretory responses to capsaicin, electrical field stimulation, and the neurokinin-1 (NK-1) agonist [Sar(9),Met(O(2))(11)]substance P were examined in Ussing chambers. Sangre de grado facilitated the healing of experimental gastric ulcer, reducing myeloperoxidase activity, ulcer size, and bacterial content of the ulcer. The expression of proinflammatory genes tumor necrosis factor-alpha, inducible nitric oxide synthase (iNOS), interleukin (IL)-1beta, IL-6, and cyclooxygenase-2 was upregulated by ulcer induction but reduced by sangre de grado treatment, particularly iNOS and IL-6. In Ussing chambers, sangre de grado impaired the secretory response to capsaicin but not to electrical field stimulation or the NK-1 agonist. We conclude that sangre de grado is a potent, cost-effective treatment for gastrointestinal ulcers and distress via antimicrobial, anti-inflammatory, and sensory afferent-dependent actions.
Assuntos
Diarreia/tratamento farmacológico , Fitoterapia , Úlcera Gástrica/tratamento farmacológico , Animais , Capsaicina/farmacologia , Ciclo-Oxigenase 2 , Primers do DNA , Cultura em Câmaras de Difusão , Relação Dose-Resposta a Droga , Estimulação Elétrica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cobaias , Íleo/efeitos dos fármacos , Íleo/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Neurônios Aferentes/fisiologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Peru , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1/agonistas , Substância P/análogos & derivados , Substância P/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Exposure of the abdomino-pelvic region to ionizing radiation, such as that received during radiotherapy, is associated with the development of a number of untoward symptoms which may limit the course of therapy or which may involve serious chronic intestinal disease. While the mucosal dysfunction surrounding acute radiation enteritis is generally ascribed to the effects of ionizing radiation on the cell cycle of epithelial stem cells of the intestinal crypts and subsequent epithelial loss, recent evidence suggests that other, earlier events also play a role. The severity of these early events may determine the incidence and severity of chronic enteritis. The mechanism for this is unclear, but may relate to radiation-induced compromise of host defence responses to luminal pathogens or antigens. This review will address the current state of knowledge of the pathogenesis of radiation-induced intestinal dysfunction, focusing on events which occur in the mucosa, and will discuss what the future may hold with respect to the treatment of radiation-associated diseases of the intestinal tract.
Assuntos
Enterite/etiologia , Mucosa Intestinal/efeitos da radiação , Lesões por Radiação/etiologia , Radioterapia/efeitos adversos , Ciclo Celular , Doença Crônica , Células Epiteliais/citologia , Células Epiteliais/efeitos da radiação , Humanos , Mucosa Intestinal/patologiaRESUMO
The relative contributions of cyclooxygenase (COX)-1 and COX-2 in mediating prostaglandin (PG)-dependent chloride secretion were investigated in segments of mouse colon mounted in Ussing-type diffusion chambers. COX-2 mRNA and protein were constitutively expressed as shown by reverse transcription-polymerase chain reaction and Western immunoblot, respectively. COX-2 immunoreactivity was detected immunohistochemically in cells lying subjacent to the crypt epithelial cells. In segments of colon mounted in Ussing chambers, arachidonic acid caused a concentration-dependent increase in short-circuit current that was blocked by piroxicam, the COX-2 inhibitor NS-398, and the COX-1 inhibitor SC-560. Exposure to the PG-dependent secretagogue, bradykinin, also caused an increase in short-circuit current that was not blocked by piroxicam or SC-560, and only by the highest dose of NS-398. When incubated in the presence of 10 microM arachidonic acid, segments of mouse colon produced both PGE(2) and PGD(2). Synthesis of PGE(2) but not PGD(2) was blocked by NS-398 and SC-560. These data demonstrate that both COX-1 and COX-2 are constitutively expressed in the mouse colon, and both contribute to PG-dependent electrolyte transport.
Assuntos
Colo/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/fisiologia , Animais , Western Blotting , Cloretos/metabolismo , Colo/citologia , Colo/efeitos dos fármacos , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Eletrólitos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Imuno-Histoquímica , Isoenzimas/biossíntese , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Pirazóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologiaRESUMO
Ionizing radiation induces intestinal epithelial hyporesponsiveness to secretagogues through an unknown mechanism. We investigated the role of the inducible isoform of nitric oxide (NO) synthase (iNOS)-derived NO in radiation-induced hyporesponsiveness. C57BL/6 mice were sham treated or exposed to 10-Gy gamma-radiation and were studied 3 days later. Tissues were mounted in Ussing-type diffusion chambers to assess chloride secretion in response to electrical field stimulation (EFS) and forskolin (10 microM). Transport studies were also repeated in iNOS-deficient mice. White blood cell counts were significantly lower in irradiated mice, and there was no inflammatory response as shown by myeloperoxidase activity and histological assessment. iNOS mRNA levels and nitrate/nitrite concentrations were significantly elevated in irradiated colons. iNOS immunoreactivity localized to the epithelium. Colons from irradiated wild-type, but not iNOS-deficient, mice exhibited a significant reduction in the responsiveness of the tissue to EFS and forskolin. The hyporesponsiveness was reversed by L-N(6)-(1-iminoethyl)lysine, 1400W, and dexamethasone treatments. iNOS-derived NO mediates colonic hyporesponsiveness 3 days after irradiation in the mouse in the absence of an inflammatory response.