The induction of intestinal inflammation as a result of abdominal surgery is an essential factor in postoperative ileus (POI) development. Electroacupuncture (EA) at ST36 has been demonstrated to relieve intestinal inflammation and restore gastrointestinal dysmotility in POI. This study aims to elucidate the neuroimmune pathway involved in the anti-inflammatory properties of EA in POI.

After intestinal manipulation (IM) was performed to induce POI, intestinal inflammation and motility were assessed 24 h post-IM, by evaluating gastrointestinal transit (GIT), cytokines expression, and leukocyte infiltration. Experimental surgery, pharmacological intervention, and genetic knockout mice were used to elucidate the neuroimmune mechanisms of EA.

EA at ST36 significantly improved GIT and reduced the expression of pro-inflammatory cytokines and leukocyte infiltration in the intestinal muscularis following IM in mice. The anti-inflammatory effectiveness of EA treatment was abolished by sub-diaphragmatic vagotomy, whereas splenectomy did not hinder the anti-inflammatory benefits of EA treatment. The hexamethonium chloride (HEX) administration contributes to a notable reduction in the EA capacity to suppress inflammation and enhance motility dysfunction, and EA is ineffective in α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice.

EA at ST36 prevents intestinal inflammation and dysmotility through a neural circuit that requires vagal innervation but is independent of the spleen. Further findings revealed that the process involves enteric neurons mediating the vagal signal and requires the presence of α7nAChR. These findings suggest that utilizing EA at ST36 may represent a possible therapeutic approach for POI and other immune-related gastrointestinal diseases.

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂ O₂ ) contribute to epithelial damage and ion transport dysfunction (key events in inflammatory diarrhoea) in inflammatory bowel disease (IBD). The aim of this study was to identify if H₂ O₂ mediates suppression of colonic ion transport function in the murine dextran sulfate sodium (DSS) colitis model by using the H₂ O₂ degrading enzyme, catalase. Colitis was induced by administering DSS (4%) in drinking water for 5 days followed by 3 days on normal H₂ O. Mice were administered either pegylated catalase or saline at day -1, 0 and +1 of DSS treatment. Ion transport responses to the Ca²⁺ -dependent agonist, carbachol (CCh), or the cAMP-dependent agonist, forskolin, were measured across distal colonic mucosa mounted in Ussing chambers. Parameters of DSS-induced inflammation (loss in body weight, decreased colon length, altered stool consistency), were only partially alleviated by catalase while histology was only minimally improved. However, catalase significantly reversed the DSS-induced reduction in baseline ion transport as well as colonic I_sc responses to CCh. However, ion transport responses to forskolin were not significantly restored. Catalase also reduced activation of ERK MAP kinase in the setting of colitis, and increased expression of the Na⁺ -K⁺ -2Cl^- cotransporter, NKCC1, consistent with restoration of ion transport function. Ex vivo treatment of inflamed colonic mucosae with catalase also partially restored ion transport function. Therefore, catalase partially prevents, and rescues, the loss of ion transport properties in DSS colitis even in the setting of unresolved tissue inflammation. These findings indicate a prominent role for ROS in ion transport dysfunction in colitis and may suggest novel strategies for the treatment of inflammatory diarrhoea.

Acid-sensing pathways, which trigger mucosal defense mechanisms in response to luminal acid, involve the rapid afferent-mediated "capsaicin pathway" and the sustained "prostaglandin (PG) pathway." Luminal acid quickly increases protective PG synthesis and release from epithelia, although the mechanism by which luminal acid induces PG synthesis is still mostly unknown. Acid exposure augments purinergic ATP-P2Y signaling by inhibition of intestinal alkaline phosphatase activity. Since P2Y activation increases intracellular Ca2+, we further hypothesized that ATP-P2Y signals increase the generation of H2O2 derived from dual oxidase, a member of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family activated by Ca2+. Our recent studies suggest that acid exposure increases H2O2 output, followed by phospholipase A2 and cyclooxygenase activation, increasing PG synthesis. Released prostaglandin E2 augments protective HCO3- and mucus secretion via EP4 receptor activation. Thus, the PG pathway as a component of duodenal acid sensing consists of acid-related intestinal alkaline phosphatase inhibition, ATP-P2Y signals, dual oxidase 2-derived H2O2 production, phospholipase A2 activation, prostaglandin E2 synthesis, and EP4 receptor activation. The PG pathway is also involved in luminal bacterial sensing in the duodenum via activation of pattern recognition receptors, including Toll-like receptors and nucleotide-binding oligomerization domain 2. The presence of acute mucosal responses to luminal bacteria suggests that the duodenum is important for host defenses and may reduce bacterial loading to the hindgut using H2O2, complementing gastric acidity and anti-bacterial bile acids.

Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies.

H(2)O(2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H(2)O(2) inhibits NaCl absorption and activates Cl(-) secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl(-)/OH(-) (HCO(3)(-)). The present studies were, therefore, undertaken to examine the direct effects of H(2)O(2) on OH(-) gradient-driven DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate)-sensitive (36)Cl(-) uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H(2)O(2) (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl(-)/OH(-) exchange activity in Caco-2 cells. H(2)O(2)-mediated inhibition of Cl(-)/OH(-) exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H(2)O(2) also induced phosphorylation of Fyn and p85 (the regulatory subunit of PI3K) in Caco-2 cells. Moreover, an increased association of Fyn and p85 was observed in response to H(2)O(2), resulting in the activation of the downstream target PLCgamma1 (phospholipase Cgamma1). Elevated intracellular Ca(2+) levels and PKCalpha (protein kinase Calpha) functioned as downstream effectors of H(2)O(2)-induced PLCgamma1 activation. Our results, for the first time, provide evidence for H(2)O(2)-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCgamma1 and Ca(2+)-dependent PKCalpha, resulting in the inhibition of Cl(-)/OH(-) exchange activity. These findings suggest that H(2)O(2)-induced inhibition of the Cl(-)/OH(-) exchange process may play an important role in the pathophysiology of diarrhoea associated with inflammatory disorders, where the amount of reactive oxygen species is markedly elevated.

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H(2)O(2)), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl(-) secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H(2)O(2) inhibit Ca(2+)-dependent Cl(-) secretion across T(84) colonic epithelial cells by elevating cytosolic Ca(2+), which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca(2+)-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca(2+)-dependent stimuli is mediated in part by K(+) efflux through basolateral K(+) channels and Cl(-) uptake by the Na(+)-K(+)-2Cl(-) cotransporter, NKCC1. We demonstrate that H(2)O(2) inhibits Ca(2+)-dependent basolateral K(+) efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl(-) conductance. Thus, we have demonstrated that H(2)O(2) inhibits Ca(2+)-dependent Cl(-) secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.

Stressful life events and infections contribute to gut disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). We used a pig model to analyse whether this could be linked to altered mediator sensitivity of the epithelial lining.

Uninfected control pigs or pigs with subclinical Salmonella (S.) typhimurium DT 104 infection were killed either without (ConRest, InfRest) or with prior 8-h transportation (ConTrans, InfTrans). Short-circuit current (I(sc)), tissue conductance (G(t)) and release of mast cell mediators were monitored in isolated colonic epithelia mounted in Ussing chambers. Epithelia were exposed to histamine (100 microM, mucosally), substance P (SP; 1 microM, serosally), calcimycin A23187 (1 microM, serosally) and theophylline (10 mM, bilaterally). Transepithelial flux of histamine and colonic activities of histamine N-methyltransferase (HMT) and diamine oxidase (DAO) were determined.

S. infection decreased baseline I(sc), G(t) and histamine fluxes, while transportation had no effect on these values. Mucosal histamine increased I(sc) only in ConTrans pigs. This was not associated with increased mucosal-to-serosal flux of histamine but with a 2-fold increased DAO activity. Serosal SP increased I(sc) only in transported animals, but the increase was six times higher in ConTrans versus InfTrans pigs. Effectiveness of SP was not dependent on the release of histamine or prostaglandin D2. A23187 and theophylline elicited increases in I(sc) that were not different between treatments.

Transportation stress facilitates secretory responses of the colonic epithelium to SP and luminal histamine. This is suppressed by subclinical S. infection. Effects of S. infection on porcine colon resemble, in part, the known effects of an oral S. endotoxin application.

To determine effects of reactive oxygen metabolites (ROMs), with and without flunixin meglumine, on equine right ventral colon (RVC) in vitro.

18 healthy horses and ponies.

In 3 groups of 6 animals each, short-circuit current and conductance were measured in RVC mucosa in Ussing chambers. The 3 groups received physiologic saline (0.9% NaCl) solution, IV, 10 minutes before euthanasia and tissue incubation in Krebs-Ringer-bicarbonate (KRB) solution; flunixin meglumine (1.1 mg/kg, IV) 10 minutes before euthanasia and tissue incubation in KRB solution; or physiologic saline solution, IV, 10 minutes before euthanasia and incubation in KRB solution with 2.7 x 10(5)M flunixin meglumine. Incubation conditions included control (no addition) and ROM systems, including addition of 1 mM xanthine and 80 mU of xanthine oxidase (to produce the superoxide radical), 1 mM H(2)O(2), and 1 mM H(2)O(2) and 0.5 mM ferrous sulfate (to produce the hydroxyl radical).

All ROMs that were added or generated significantly increased the short-circuit current except in tissues coincubated with flunixin meglumine, and they induced mild epithelial vacuolation and apoptosis, but did not disrupt the epithelium nor change conductance, lactate dehydrogenase release, or [(3)H]mannitol flux.

Responses to ROMs could be attributed to increased chloride secretion and inhibited neutral NaCl absorption in equine RVC, possibly by stimulating prostaglandin production. The ROMs examined under conditions of this study could play a role in prostaglandin-mediated colonic secretion in horses with enterocolitis without causing direct mucosal injury.

To investigate the effects of luminal exposure to H2O(2) and two related thiol oxidizing agents on basal and stimulated chloride secretion in native colon using electrophysiological and pharmacological approaches.

Unstripped rat distal colon segments were mounted in Ussing chambers. Potential difference, calculated resistance and short-circuit current across unstripped colon segments were monitored with a dual voltage/current clamp. Paracellular permeability was assessed by measuring the mucosa-to-serosa flux of a fluorescent probe (FITC).

Luminal exposure to hydrogen peroxide transitorily stimulated chloride secretion without altering barrier function. This stimulatory effect could be blocked by basolateral atropine but not indomethacin. The cysteine and methionine oxidizing compounds, phenylarsine oxide and chloramine T respectively, mimicked the effect of H2O(2), except for a drop in transcolonic resistance after 30 min. In contrast to the observed stimulatory effect on basal secretion, cAMP-stimulated electrogenic ion transport was blunted by luminal H2O(2). However, the Ca(2+)-activated response remained unchanged.

H2O(2) may be an important selective modulator of intestinal ion and water secretion in certain pathologic conditions such as inflammation or ischemia-reperfusion by multiple mechanisms.

The goal of this study was to determine the effects of peroxynitrite (ONOO-) on smooth muscle membrane potential and vasomotor function in rabbit carotid arteries. ONOO- is known to affect vascular tone by several mechanisms, including effects on K+ channels. Xanthine (X, 0.1 mM), xanthine oxidase (XO, 0.01 U/ml), and a low concentration of sodium nitroprusside (SNP, 10 nM) were used to generate ONOO-. In the common carotid artery, X and XO (X/XO) in the presence of SNP tended to increase tension. In contrast, in the internal carotid artery, X/XO in the presence of SNP transiently hyperpolarized the membrane (-8.5 +/- 1.8 mV, mean +/- SE) and decreased tension (by 85 +/- 5.6%). In internal carotid arteries, in the absence of SNP, X/XO did not hyperpolarize the membrane and produced much less relaxation (by 23 +/- 5.6%) than X/XO and SNP. Ebselen (50 microM) inhibited both hyperpolarization and relaxation to X/XO and SNP, and uric acid (100 microM) inhibited relaxation. Glibenclamide (1 microM) abolished hyperpolarization and inhibited relaxation during X/XO and SNP. Charybdotoxin (100 nM) or tetraethylammonium (1 mM) did not affect hyperpolarization or relaxation, respectively. These results suggest that ONOO- hyperpolarizes and relaxes smooth muscle in rabbit internal carotid artery but not in common carotid artery through activation of K(ATP) channels.

The eye is a unique organ because of its constant exposure to radiation, atmospheric oxygen, environmental chemicals and physical abrasion. That oxidative stress mechanisms in ocular tissues have been hypothesized to play a role in diseases such as glaucoma, cataract, uveitis, retrolental fibroplasias, age-related macular degeneration and various forms of retinopathy provides an opportunity for new approaches to their prevention and treatment, In the anterior uvea, both H2O2 and synthetic peroxides exert pharmacological/toxicological actions tissues of the anterior uvea especially on the sympathetic nerves and smooth muscles of the iris-ciliary bodies of several mammalian species. Effects produced by peroxides require the presence of trace amounts of extracellular calcium and the functional integrity of mitochondrial calcium stores. Arachidonic acid metabolites appear to be involved in both the excitatory action of peroxides on sympathetic neurotransmission and their inhibitory effect on contractility of the iris smooth muscle to muscarinic receptor activation. In addition to the peroxides, isoprostanes (products of free radical catalyzed peroxidation of arachidonic acid independent of the cyclo-oxygenase enzyme) can also alter sympathetic neurotransmission in anterior uveal tissues. In the retina, both H2O2 and synthetic peroxides produced an inhibitory action on potassium depolarization induced release of [3H] D-aspartate, in vitro and on the endogenous glutamate and glycine concentrations in vivo. Effects caused by peroxides in the retina are mediated, at least in part, by second messengers such as nitric oxide, prostaglandins and isoprostanes. The ability of H2O2 to alter the integrity of neurotransmitter pools from sympathetic nerves in the anterior uvea and glutaminergic nerves in the retina could underlie its role in the etiology of glaucoma.

H2O2 and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2 and damage functions of membrane proteins except for PGE2 receptors. The aim of this study was to examine whether the preserved PGE2 actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2 prevented the inhibition of H2O2 or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2 and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2 pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2 and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2 before H2O2 and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2 prevents the muscle cell damage caused by H2O2 and TCDC due to the resistance of PGE2 receptors to agonist-induced desensitization. The preservation of PGE2 receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.

Intestinal epithelial cell function is compromised by local immune and inflammatory responses. In this study, we examined the possibility that intestinal epithelial cell injury occurs in the presence of activated inflammatory cells, such as neutrophils and macrophages, via production of reactive oxygen species (ROS). Following exposure to 50-150 microM H2O2 levels of mRNA and protein for Fas and, to a lesser degree, Fas-L were increased and intestinal epithelial cells underwent apoptosis. Treatment of H2O2-exposed cells with agonistic anti-Fas antibody, but not isotype control antibody, significantly enhanced apoptosis. Apoptosis was associated with the activation of caspase 8, while Z-IETD, an inhibitor of caspase 8, blocked apoptosis of H2O2-exposed intestinal epithelial cells. Thus, ROS induced Fas and Fas-L expression in association with intestinal epithelial cell apoptosis. These data support the hypothesis that, following exposure to oxidative stress, enterocytes are primed for cell death via Fas-mediated pathways.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of muscle dysfunction in acute inflammatory processes. The aim of these studies was to determine the effects of ROS on gallbladder muscle function in vitro. Single muscle cells were obtained by enzymatic digestion. H(2)O(2) (70 microM) caused maximal contraction of up to 14% and blocked the response to CCK-8, ACh, and KCl. It did not affect the contractions induced by guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and inositol 1,4,5-trisphosphate that circumvent membrane receptors. The contraction induced by H(2)O(2) was inhibited by AACOCF(3) [cytosolic phospholipase A(2) (cPLA(2)) inhibitor], indomethacin (cyclooxygenase inhibitor), chelerythrine [protein kinase C (PKC) inhibitor], or PD-98059 [mitogen-activated protein kinase (MAPK) inhibitor]. H(2)O(2) also reduced the CCK receptor binding capacity from 0.36 +/- 0.05 pmol/mg protein (controls) to 0.17 +/- 0.03 pmol/mg protein. The level of lipid peroxidation as well as the PGE(2) content was significantly increased after H(2)O(2) pretreatment. Unlike superoxide dismutase, the free radical scavenger catalase prevented the H(2)O(2) induced contraction, and its inhibition of the CCK-8 induced contraction. It is concluded that ROS cause damage to the plasma membrane of the gallbladder muscle and contraction through the generation of PGE(2) induced by cPLA(2)-cyclooxygenase and probably mediated by the PKC-MAPK pathway.

Interleukin 1beta induced both nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2) gene expression in dorsal root ganglion explant culture with increased NOS-2 and COX-2 activities, and corresponding increases in the production of nitric oxide and prostaglandin E(2). The proinflammatory cytokine also increased 8-isoprostaglandin F(2alpha) concentration, an index of oxidant stress-mediated production of lipid hydroperoxides/reactive oxygen species. The signaling mechanisms by which interleukin 1beta regulates NOS-2 and COX-2 genes remain obscure. Reactive oxygen species play an important role in inflammatory processes as mediators of injury, and potentially as intracellular signaling molecules in interleukin 1beta-mediated regulation of gene expression. The effects of antioxidants that act by different mechanisms on interleukin 1beta-mediated NOS-2 and COX-2 gene expression were studied in rat dorsal root ganglion explants. The oxidant scavenger pyrrolidine dithiocarbamate abolished interleukin 1beta-induced NOS-2 mRNA accumulation and decreased nitric oxide production in a concentration-dependent manner, thus indicating that this antioxidant decreased either the transcription of NOS-2 gene or the stability of NOS-2 mRNA. In contrast, pyrrolidine dithiocarbamate significantly inhibited COX-2 gene expression at the posttranscriptional level, since pyrrolidine dithiocarbamate did not affect interleukin 1beta-induced COX-2 mRNA transcripts but inhibited COX-2 protein expression and prostaglandin E(2) production. Rotenone, another antioxidant that attenuates reactive oxygen species production by inhibiting the mitochondrial electron transport system, failed to inhibit interleukin 1beta-induced NOS-2 and COX-2 mRNA-encoding transcripts. However, rotenone inhibited NOS-2 and COX-2 proteins and associated nitric oxide and prostaglandin E(2) production, respectively, suggesting a posttranscriptional target for interleukin 1beta-mediated regulation of NOS-2 and COX-2 gene expression. Furthermore, both pyrrolidine dithiocarbamate and rotenone also decreased interleukin 1beta-induced 8-isoprostaglandin F(2alpha) production. These results indicate that not only transcriptional regulation, but also posttranscriptional events are involved in a redox-sensitive regulation of interleukin 1beta-induced NOS-2 and COX-2 gene expression in the dorsal root ganglia. Overall, interleukin 1beta-induced oxidant stress appears to regulate NOS-2 and COX-2 gene expression primarily at the level of protein translation. By implicating reactive oxygen species production in interleukin-1beta receptor-activated molecular signaling in the dorsal root ganglia, our data suggest a possible novel target for intervention in cytokine-mediated inflammatory processes.

The present study was undertaken to investigate the effect of reactive oxygen species on prostaglandin F2 alpha (PGF2 alpha) production by human endometrial stromal cells (ESC).

Isolated ESC were incubated with hydrogen peroxide, which induces lipid peroxidation. Hydrogen peroxide increased both intracellular and medium concentrations of PGF2 alpha (P < 0.01). A time course study showed that hydrogen peroxide significantly increased PGF2 alpha concentrations in the medium after 6 h incubation (P < 0.01), after which no further increase was observed. To study whether the increase in PGF2 alpha production caused by hydrogen peroxide was mediated by cyclooxygenase, ESC were incubated with indomethacin (0.5 microg/ml), an inhibitor of cyclooxygenase, in the presence of hydrogen peroxide. Indomethacin significantly blocked the increases in PGF2 alpha production caused by hydrogen peroxide (P < 0.01). Hydrogen peroxide also increased PGF2 alpha production by decidualized ESC (P < 0.01), induced by the incubation with medroxyprogesterone acetate (10(-6) mol/l) and oestradiol (10(-8) mol/l).

Reactive oxygen species stimulate PGF2 alpha production in ESC, suggesting that they might influence endometrial function by regulating PGF2 alpha production.

Muscle strips from experimental acute cholecystitis (AC) exhibit a defective contraction. The mechanisms responsible for this impaired contraction are not known. The present studies investigated the nature of these abnormalities. AC was induced by ligating the common bile duct of guinea pigs for 3 days. Contraction was studied in enzymatic dissociated muscle cells. Cholecystokinin (CCK) and prostaglandin E2 (PGE2) receptor binding studies were performed by radioreceptor assay. The levels of lipid peroxidation, cholesterol, phospholipid, and H2O2 as well as the catalase and superoxide dismutase (SOD) activities were determined. PGE2 content was measured by radioimmunoassay. Muscle contraction induced by CCK, ACh, or KCl was significantly reduced in AC, but PGE2-induced contraction remained normal. GTPgammaS, diacyglycerol (DAG), and 1,4,5-trisphosphate (IP3), which bypass the plasma membrane, caused a normal contraction in AC. The number of functional receptors for CCK was significantly decreased, whereas those for PGE2 remained unchanged in AC. There was a reduction in the phospholipid content and increase in the level of lipid peroxidation as well as H2O2 content in the plasma membrane in AC. The PGE2 content and the activities of catalase and SOD were also elevated. These data suggest that AC cause damage to the constituents of the plasma membrane of muscle cells. The preservation of the PGE2 receptors may be the result of muscle cytoprotection.

There is increasing evidence supporting the involvement of immune cells and mediators in the control of intestinal physiology. Cell coculture systems and epithelial cell lines have provided convenient model systems for the study of immunomodulation of epithelial function. Abundant cytokines and immune mediators have been shown to directly or indirectly alter epithelial transport of ions and macromolecules. Animal models of hypersensitivity have shown that luminal antigen challenge in the intestine of sensitized rats induces a rapid ion secretory response due to enhanced transepithelial transport of antigen. Transport of ions and macromolecules is highly regulated and an important component of host defense. Dysregulation of epithelial function may play a role in several intestinal disorders, such as inflammatory bowel diseases and food allergy.

Diarrhea is one of the major complications of inflammatory bowel disease. The role of oxidants in promoting net intestinal secretion is important, but the cellular mechanisms underlying their effects are unclear. We examined the effects and defined the cellular actions of the oxidant monochloramine (NH(2)Cl) on anion secretion in human colonic T84 cells.

Effects of NH(2)Cl on basal and agonist-stimulated short-circuit current (Isc) of T84 monolayers were determined. Apical Cl(-) and basolateral K(+) conductances were measured by efflux of (125)I(-) and (86)Rb(+), respectively.

NH(2)Cl alone had little effect on Isc and (125)I(-) efflux. However, pretreatment with NH(2)Cl led to a concentration-dependent potentiation of the Ca(2+)-mediated Isc and of submaximal cAMP-mediated responses. These effects were associated with increased basolateral K(+) channel conductance and were blocked by increasing cellular Ca(2+) buffering capacity with Quin-2. Whole-cell voltage clamp experiments showed that NH(2)Cl potentiated Ca(2+) activation of basolateral K(+) channel conductance.

Oxidants potentiate both Ca(2+)- and cAMP-stimulated Cl(-) secretion by a direct effect on calcium-activated basolateral K(+) channel conductance, lowering its Ca(2+) activation threshold. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.

We have investigated the effects of hydrogen peroxide (H(2)O(2)), a potent naturally occurring oxidant on cell signaling and viability in the pluripotent HT29 intestinal cell line. There was a dose-dependent reduction in cell viability upon exposure to H(2)O(2) as measured by the XTT assay. Features of apoptosis were indicated by the findings of PARP and caspase 3 cleavage, as well as changes in cell morphology using phase contrast and nuclear fragmentation using fluorescence microscopy. There was a dose-dependent increase in the activation of p45-JNK, p42/p44-ERK, and p38-HOG. Surprisingly, oxidant-induced cell injury could be attenuated by preincubation with PD98059 to 50% of untreated control cells (P = 0.002). This and UO126, another MEK inhibitor were ably to reproducibly inhibit p45-JNK activation induced by hydrogen peroxide. Transfection with kinase-inactive constructs of JNK and ERK revealed that the improvement in cell viability was due to inhibition of JNK and not ERK. Transient transfections with AP-1 and NF-kappaB luciferase reporter constructs did not reveal any transcriptional activation due to hydrogen peroxide exposure however, in both cases the basal levels of transcriptional activity were suppressed in the presence of PD98059. It is concluded that JNK mediates H(2)O(2)-induced cellular injury in the HT29 cell line, and additionally, we report for the first time that JNK activation can be inhibited by both PD98059 and UO126 at conventional doses used to inhibit MEK.

During the past three decades, many studies have been conducted to determine the precise role of eicosanoids in colorectal physiology and pathophysiology. This research has increased our understanding of bioactive lipid signaling, and may contribute to the development of more effective therapeutic modalities for digestive diseases in the future. The purpose of this report is to provide a brief overview of the role of eicosanoids in the colon and rectum. This information has been organized according to both functional and disease-related categories. The role of eicosanoids in colonic secretion, motility, inflammatory bowel disease, and colorectal neoplasia will be discussed.

In the present study, we investigated the effect of inhibition of cyclooxygenase (COX) with flurbiprofen (FBF) on peroxide-induced enhancement of field-stimulated [3H]norepinephrine ([3H]NE) release from bovine isolated irides. Furthermore, the effect of FBF was examined on peroxide-induced attenuation of contractions evoked by carbachol on this tissue. Irides were prepared for studies of neurotransmitter release and for measurement of contractile tension in vitro. Pretreatment of tissues with FBF (10 microM) caused significant (P < 0.001) rightward shifts of concentration-response curves to H2O2 and also decreased cumene hydroperoxide (cuOOH)-induced enhancement of evoked [3H]NE release. FBF (10 microM) partially prevented the attenuation of carbachol-induced contractions induced by H2O2 (300 microM) and cuOOH (300 microM). We conclude that inhibition of the biosynthesis of prostanoids reduced both the prejunctional stimulatory effects of H2O2 and cuOOH on sympathetic neurotransmission and inhibitory effects of peroxides on carbachol-induced contractions the in the bovine isolated iris.

Diseases of the gastrointestinal tract that require pharmacologic management, usually in combination with other treatments, are gastric ulcers (omeprazole and others), colic (laxatives, analgesics), diarrhea (antibiotics, protectants and absorbents, glucocorticoids, motility inhibitors), reperfusion injury, postoperative ileus (prokinetic drugs), and adhesions. There is growing evidence that nonsteroidal anti-inflammatory drugs can alter important physiologic properties of the intestine; however, these drugs are valuable analgesics for horses and their use should be tempered with an awareness of their harmful effects. The role of antibiotics in treating gastrointestinal disease is controversial, but their ability to induce life-threatening diarrhea is well known and invites caution and defensible use of these drugs in horses.

The hypothesis that neuroepithelial endocrine (NEE) cells control spontaneous tone in isolated guinea pig tracheal preparations was examined. Epithelium-denuded preparations were unable to develop a normal oscillating tone in 12% oxygen (corresponding to systemic arterial oxygen levels) and, instead, developed a strong, smooth tone, similar to the "classic" tone in 94% oxygen. Inhibition of the hydrogen peroxide-producing NADPH oxidase in the NEE cells by 20 microM diphenyleneiodonium chloride transformed, in intact preparations in 94% oxygen, the tone from a strong, smooth type to an oscillating tone of considerably less force. Similar experiments in denuded preparations showed no change of tone and no oscillations. After pretreatment with the catalase inhibitor 3-amino-1,2, 4-triazole (1 mM), addition of 2 mM hydrogen peroxide to intact preparations displaying the oscillating tone caused a transformation to a strong, smooth type. These findings support the hypothesis that the spontaneous tone in this preparation is largely controlled by the oxygen-sensing NEE cells. For the first time, previous findings on isolated cells can be linked to effects in intact tissue preparations. The results also suggest that the regulation by the NEE cells involves the release of powerful relaxing and contracting factors from the epithelium.

By considering the pathophysiologic basis of inflammatory bowel diseases, a role for excessive lipid peroxidation caused by oxygen free radical compounds has been proposed repeatedly. However, to date only a few studies are available on this topic in human beings. This study was designed to assess breath alkanes in a group of patients with active inflammatory bowel disease by a technique that clearly distinguishes pentane from isoprene, to prevent overestimation of values as in previous studies.

Twenty patients with a diagnosis of active inflammatory bowel disease (10 with Crohn's disease and 10 with ulcerative colitis) were studied. Extension of the disease was similar between patient groups, and all were treated with equivalent doses of steroids and salicylates.

Breath alkanes determination was performed by a standard procedure involving a gas chromatography column able to separate pentane from isoprene.

Overall, significant differences between patients with inflammatory bowel diseases and controls were found for ethane, propane, and pentane, but not for butane and isoprene. Isoprene was clearly distinguished from pentane, demonstrating that the significant elevation of pentane levels in patients with inflammatory bowel diseases is a real phenomenon and not an artifact caused by coelution with isoprene.

An excess of lipid peroxidation is probably an important pathogenetic factor in inflammatory bowel diseases, and this may be assessed through a noninvasive method. Because this method previously also has been shown to be able to evaluate disease activity, it could be a useful tool for studying patients with inflammatory bowel diseases.

We examined the effects of H2O2 on Cl- secretion across human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a measure of Cl- secretion, was inhibited in a concentration-dependent fashion when monolayers were pretreated with H2O2 for 30 min (30-100% inhibition between 500 microM and 5 mM). Moreover, H2O2 inhibited 76% of the Cl- current across monolayers when the basolateral membranes were permeabilized with nystatin (200 micrograms/ml). When the apical membrane was permeabilized with amphotericin B, H2O2 inhibited the Na+ current (a measure of Na+-K+-ATPase activity) by 68% but increased the K+ current more than threefold. In addition to its effects on ion transport pathways, H2O2 also decreased intracellular ATP levels by 43%. We conclude that the principal effect of H2O2 on colonic Cl- secretion is inhibitory. This may be due to a decrease in ATP levels following H2O2 treatment, which subsequently results in an inhibition of the apical membrane Cl- conductance and basolateral membrane Na+-K+-ATPase activity. Alternatively, H2O2 may alter Cl- secretion by direct action on the transporters or alterations in signal transduction pathways.

Piglet cryptosporidiosis is characterized by intestinal villous damage and malabsorption, and by reduced NaCl absorption in response to prostaglandins (PGs), which act directly on the epithelium and indirectly through enteric nerves. We hypothesized that phagocyte-derived reactive oxygen metabolite (ROM) production contributed to PG synthesis and altered transport in inflamed ileum. Ileal mucosa from control and infected piglets was analyzed for villous height, PGE2, catalase (an endogenous antioxidant), and malondialdehyde (MDA, a by-product of lipid peroxidation) from d 2-8 after infection. The response of control ileal mucosa to exogenous ROM and infected mucosa to antioxidant treatment was also studied in tissues mounted in Ussing chambers. Increased levels of MDA on d 2 preceded increased PGE2 on d 3-4, which correlated with the acute diarrheal phase; however the most severe villous atrophy (d 8) correlated with the highest levels of catalase and MDA but low levels of PGE2. Control mucosa responded to H2O2 with indomethacin- and tetrodotoxin-sensitive transient increases in short circuit current (Isc), which were accompanied by increased tissue production of 6-keto-PGF1a, the stable metabolite of PGI2; however, no increased PGE2 production was detectable. A stable analog of PGI2, carbacyclin, mimicked the transient Isc response to H2O2; however, several antioxidants failed to alter the abnormal Isc of infected tissue. These results suggest that there is evidence of increased ROM production in cryptosporidial infection and that intestinal PG synthesis and inhibited NaCl absorption may be mediated partially by ROM in this model. Additional, cooperative factors, such as PGE2 production, however, are likely needed to induce the alterations in ion transport seen in this infection.

L-Arginine has been shown to induce fluid secretion in human jejunum. Nitric oxide, a derivative of L-arginine is thought to have an important role as an intestinal secretagogue.

To determine the effect of L-arginine and the nitric oxide synthase inhibitor, nitro L-arginine methyl ester (L-NAME), on fluid and electrolyte movement in rat jejunum.

A 25 cm segment of rat jejunum was perfused in situ with iso-osmotic solutions containing either (1) saline, (2) D-arginine 20, (3) L-arginine 20, (4) L-NAME 0.1, 1, or 20 mmol/l, or (5) a combination of L-arginine 20 and L-NAME 0.1, 1, or 20 mmol/l. In further groups the effect of a subcutaneous injection of L-NAME 100 mg/kg was examined in rats pretreated with either D-or L-arginine 500 mg/kg.

L-Arginine, unlike D-arginine, induced fluid secretion despite being better absorbed (mean -7.3 v 17.0 microliters/min/g; p < 0.01). L-NAME at 0.1 mmol/l had no effect on basal fluid movement but reversed L-arginine induced secretion (7.8; p < 0.05). L-NAME at 1 and 20 mmol/l induced fluid secretion (-15.4 and -28.4, respectively), which was enhanced by the addition of L-arginine (-30.0 and -41.0, respectively; both p < 0.05). A subcutaneous injection of L-NAME resulted in marked fluid secretion (-39.9) and histological evidence of intestinal ischaemia. These changes were attenuated or reversed by pretreatment with subcutaneous L- but not D-arginine.

L-arginine induces intestinal fluid secretion through production of nitric oxide. There is a delicate balance between the effect of nitric oxide as a secretagogue and its effect on maintaining blood flow and thus preventing intestinal ischaemia.

Cyclic AMP-dependent Cl- secretion is the major secretion pathway in human intestine. The aim of the present study was to examine mechanisms involved in cAMP-dependent anion secretion in human small and large intestine. Surgical resection specimens from both jejunum and distal colon were studied under short circuited conditions. Addition of the phosphodiesterase inhibitor IBMX induced an increase in the short-circuit current (Isc) equivalent to the net increase in Cl- secretion. The Isc was inhibited by diphenylamine decarboxylate (DPC; Cl- channel blocker), bumetanide (basolateral Na+/K+/2Cl- cotransporter), BaCl2 (basolateral K+ channel) and Cl- free buffer in both segments and indomethacin (cyclo-oxygenase inhibitor) in colon alone. Diphenylamine decarboxylate appears to directly inhibit secretion in jejunum, although its inhibitory effect is possibly mediated by inhibition of cyclo-oxygenase in the colon. A small component of IBMX-stimulated Isc was inhibited by acetazolamide. Cyclic AMP-dependent secretion is largely apical Cl- secretion, although a small component appears to be HCO3. Secretion is dependent on basolateral K+ channels and Na+/K+/2Cl- cotransporters and, in the colon, is inhibited by indomethacin, implying a role for cyclo-oxygenase metabolites. The chloride channel blocker DPC inhibits secretion in both areas. This class of compounds may have potential for treatment of secretory diarrhoea.

This study examined whether the immunocyte recruitment associated with a mild inflammatory state induced by acetic acid would produce detectable sulfidopeptide leukotriene (LT) levels from colonic tissues or in dialysates. Histological examination and measurements of peroxidase activities of inflamed tissues indicated edema, hyperplasia and neutrophil infiltration. Significant elevated LTB4 and prostaglandin E2(PGE2) levels were found but only slight elevations in sulfidopeptide LTs occurred. A slight elevation in eosinophil peroxidase indicated that eosinophil infiltration also occurred. The increase in sulfidopeptide LT levels appeared insufficient by itself to alter secretory responses in the distal colon. However, combined with other immunocyte products such as PGs, the sulfidopeptide LTs may influence the symptomology of inflammatory bowel disease.

The colonic lumen is likely to contain oxidants derived from unabsorbed dietary materials including transition metals, rancid fat, drugs and bacterial metabolites. The present study looks at the effect of luminal exposure of different oxidants on colonic mucosal lipid peroxidation and absorptive function. All the oxidants tested induced fluid and electrolyte secretion and indomethacin, a prostaglandin synthesis inhibitor reversed this effect. Oxidants did not induce mucosal lipid peroxidation. This study suggests that oxidants induce functional alterations in colon possibly through stimulation of prostaglandin generation without influencing mucosal lipid peroxidation.

C5a is a biologically active polypeptide formed during the course of complement activation and is known to possess histamine-releasing and neutrophil chemotactic properties. In the present study, we have demonstrated that C5a can regulate electrolyte transport across guinea pig ileum, and we have investigated its mechanism of action. Segments of ileum stripped of longitudinal muscle were mounted in Ussing chambers (Krebs' buffer, 37 degrees C, 95% O2/5% CO2) for monitoring short-circuit current (Isc). Serosal application of C5a evoked a transient increase in Isc with an EC50 value of 5.0 nM indicating a potent effect. The C5a-induced increase in Isc was abolished by elimination of both Cl- and HCO3- from the Krebs' solution. Pretreatment with the cyclooxygenase inhibitor indomethacin (5 microM), the neurotoxin tetrodotoxin (0.5 microM) and the H1 receptor antagonist pyrilamine (0.5 microM) reduced the effect of C5a, but the muscarinic antagonist atropine (0.5 microM) was without effect. C5a (100 nM) also evoked the release of histamine (measured by radioimmunoassay in the serosal bathing fluid) by 282% of the control value. In conclusion, in the guinea pig ileum C5a stimulates mucosal anion secretion by releasing histamine and cyclooxygenase products of arachidonic acid. The response is also mediated, in part, via non-chloinergic enteric nerves.

Determining the role of eicosanoids in gastrointestinal physiology and pathophysiology has been an active area of investigation over the past 20 years. The landmark discovery of prostaglandin endoperoxide synthase and other enzymes involved in the production of arachidonic acid products (lipoxygenases and epoxygenases) ushered in a new era of research. The goal of this review is to distill a large body of work pertaining to studies of eicosanoids in the gastrointestinal tract. This review has been organized according both to functional (secretion and motility) and disease-related (inflammation, mucosal injury, and neoplasia) effects. The aim of this article is to present a clear summary of this area of gastroenterology so that future research can be directed in a logical and productive manner.

Inflammatory mediators may contribute to the diarrhea associated with colitis. Although the secretory action of such mediators is reported in normal tissue, there is little information regarding their effects on inflamed tissue. We examined the short-circuit current response (Isc) to these mediators, in mitomycin-C (MC)-induced colitis, a model with histological similarities to colitis in man. Rats were injected once with MC (3.25 mg/kg, intraperitoneally) or vehicle. The colons were removed three and seven days later and mounted, devoid of muscularis, in Ussing chambers for measurement of Isc, potential difference (PD), and resistance (Rt). MC-treated rats had diarrhea after three days, and microscopic studies revealed colonic inflammation. There were no significant differences in Rt, PD, and Isc between control and MC-treated tissues at three and seven days. Maximal increases in Isc to bradykinin, prostaglandin E1, carbachol, substance P, and serotonin were depressed at three and/or seven days after MC. The Isc response to theophylline was not affected. Theophylline activates secretion through an intracellular mechanism; the other agonists act by interaction with epithelial cell membranes. Therefore, the mechanism for the decreased Isc may result from uncoupling of receptors to second-messenger systems or desensitization of receptor-linked secretory mechanisms.

The role of reactive oxygen species in injury to cells lining the gastrointestinal tract has gained importance in recent times, and their role in intestinal diseases has been studied.

In this study colonic epithelial cells isolated from the rat colon were exposed in vitro to various oxidants such as menadione, xanthine-xanthine oxidase, hydrogen peroxide, cumene hydroperoxide, and tertiary butyl hydroperoxide, separately. Changes in cell viability, thiol status, and the antioxidant enzyme activities were measured.

Colonocytes were found to be sensitive to menadione and were not affected by various other oxidants. Decrease in cell viability, depletion of reduced glutathione and protein thiol, and change in antioxidant enzyme activities were observed when the cells were exposed to menadione.

This study suggests that, unlike other cells, colonocytes are susceptible only to certain selective oxidants.

Reactive oxygen intermediates (ROIs) play an important role in inflammatory processes as mediators of injury and potentially in signal transduction leading to gene expression. Cyclooxygenase (COX) is a rate-limiting enzyme in prostanoid biosynthesis, and its recently cloned inducible form, COX-2, is induced by proinflammatory cytokines. This study linked ROIs to the signaling pathways that induce COX-2 expression. The hydroxyl radical scavengers DMSO (1%), as well as di- and tetramethylthiourea, inhibited IL-1-, TNF alpha-, and LPS-induced COX-2 expression in rat mesangial cells. The suppression of COX-2 mRNA expression correlated with the COX-2 protein level. In comparison with the prolonged induction of the inducible gene encoding protein-tyrosine phosphatase by hydrogen peroxide, the COX-2 gene was only transiently induced. Protein-tyrosine phosphatase is also induced by heat shock and chemical stress, whereas COX-2 is not. Superoxide was a more potent inducer for COX-2 than hydrogen peroxide. In addition, NADPH stimulated COX-2 expression, and an inhibitor of NADPH oxidase blocked COX-2 expression induced by TNF alpha. COX-2 and KC gene expression costimulated by IL-1 were inhibited differentially by the scavengers. These studies demonstrate that oxidant stress is a specific and important inducer of COX-2 gene expression. This induction may contribute to the deleterious amplification of prostanoids in inflammation and compound the direct effects of ROI production.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.

Prostaglandins stimulate electrogenic anion secretion and inhibit sodium chloride absorption in cryptosporidium induced pig diarrhoea. Because tumour necrosis factor alpha (TNF alpha) is an early mediator of inflammation and stimulates prostaglandin secretion, we investigated its effect on intestinal ion transport. Cryptosporidium infected pig ileum showed higher macrophage infiltration and tissue TNF alpha-like activity than uninfected tissues (p < 0.05, n = 4 and p < 0.05, n = 12, respectively). TNF alpha treatment of control porcine ileal mucosa increased the short circuit current (Isc), a measurement of net anion secretion in this model (p < 0.001, n = 23). This effect was blocked by 10(-6) M indomethacin and Cl- replacement. Neither acute treatment nor preincubation of colonic intestinal epithelial cell monolayers (T84) with TNF alpha stimulated the Isc. However, co-mounting of TNF alpha preincubated pig jejunal fibroblasts (P2JF) monolayers back to back with untreated T84 monolayers dose-dependently induced an indomethacin sensitive increase in Isc compared with values in untreated co-mounted monolayers (p < 0.001, n = 11). These data suggest that in infectious diarrhoea, TNF alpha may induce Cl- secretion through a paracrine mechanism involving prostaglandin release from subepithelial cells, for example fibroblasts.

Escherichia coli O157:H7 infection induces diarrhea, severe colitis, and colonic electrolyte transport abnormalities characterized by decreased Na absorption and Cl secretion. The aim of this study was to examine the role of the host inflammatory response in inducing distal colonic transport changes during infection with E. coli O157:H7.

New Zealand white rabbits aged 10 days were infected with E. coli O157:H7 strain EDL933 (plasmid+, verotoxin 1+, verotoxin 2+). Studies were performed daily from day 1 to day 5 postinfection and compared with uninfected controls (10 days old). Distal colonic ion transport was studied in vitro under short-circuited conditions in Ussing chambers, and tissue inflammation was assessed by mucosal myeloperoxidase activities and mucosal neutrophil (polymorphonuclear neutrophil [PMN]) counts. In a second study, PMN infiltration was inhibited by an anti-CD18 (leukocyte adhesion molecule) monoclonal antibody, IB4, and histology and transport were studied on day 5 postinfection.

Infection with O157:H7 induced diarrhea and inhibition of Na absorption by day 3. CI secretion occurred on day 5, coincident with tissue infiltration with PMN. Pretreatment with IB4 prevented histological damage and tissue infiltration with PMN, and it inhibited the transport abnormalities induced by infection alone.

Infection with O157:H7 reduces Na absorption and stimulates Cl secretion in the distal colon. Disruption of the epithelium and changes in colonic electrolyte transport during enterohemorrhagic E. coli are mediated by the host inflammatory response.

Hydrogen peroxide (H2O2) is a reactive oxygen species that can be produced in the digestive tract by inflammatory cells or during reperfusion following ischemia. To evaluate a possible direct effect of H2O2 on epithelial secretory cells, well-differentiated colonic T84 cells were grown to confluence on permeable membranes and studied in Ussing chambers. In this model, where the measured short-circuit current (Isc) reflects electrogenic secretion, we observed that H2O2 stimulated a concentration-dependent and transient secretory response: 5.5 mM H2O2 produced a peak Isc of 12.4 microA/cm2 after 4 min, 2.2 mM H2O2 a peak Isc of 7.9 microA/cm2 after 4 min, and 1.1 mM H2O2 a peak Isc of 5.5 microA/cm2 after 16 min (N = 5). When 97 experiments using 5.5 mM H2O2 were reviewed, the mean peak Isc response was 8.9 +/- 0.5 microA/cm2. A similar secretory response was elicited whether H2O2 was added to the serosal, to the mucosal, or simultaneously to both sides of the T84 cell monolayer. This secretory response reflected transcellular chloride secretion because it was inhibited by the depletion of chloride in the medium and by the suppression of the Na+,K+,2Cl- co-transporter activity necessary for the chloride gradient driving chloride secretion. When T84 cell monolayer resistance was studied, 5.5 mM H2O2 produced a transient decrease in resistance, reflecting transcellular chloride secretion, and a gradual decline in resistance (75% of the initial value after 55 min). The secretory response to H2O2 was increased 2-fold in T84 cells maximally stimulated with 10 nM vasoactive intestinal peptide (VIP), a neuropeptide which acts via cAMP, demonstrating synergism between the two agents. In contrast, the secretory responses produced by H2O2 and carbachol, which acts through the Ca2+ pathway, were additive. A late inhibitory effect of H2O2 was also observed: in cells previously treated with 5.5 mM H2O2, the subsequent secretory responses to either VIP or carbachol were partially inhibited. These secretory effects were specific for the oxidant properties of H2O2 because they were inhibited by 450 U/mL catalase and by 5 mM dithiothreitol, but were unaffected by 50 microM deferoxamine B or Fe3+. H2O2 may be a potential modulator of intestinal or colonic secretion in certain pathologic conditions such as inflammation or ischemia-reperfusion.