h equilibration period, the rats were injected intravenously (iv) with vehicle or drug, after which MAP, HR, MBF, MVC and ILP were recorded for a 50 rain observation period. Results: The 5-HT4 receptor agonist tegaserod (0.3 and 1.0 mg/kg iv) did not alter MBF, MVC or colonic motor activity. MAP was slightly decreased while HR did not change following treatment with tegaserod. Neither of the two 5-HT3 receptor antagonists, alosetron (0.03 and 0.1 mg/kg iv) and cflansetron (0.1 and 0.3 mg/kg iv), caused changes of MAP, HR or colonic motor activity. Both drugs, however, caused a significant and time-dependent attenuation of MBF as well as MVC. During the observation period of 35 - 50 rain, MBF and MVC were lowered by 15% and 19%, respectively, following treatment with alosetron (0.03 mg/kg iv) and reduced by 20% and 21%, respectively, following treatment with cilansetron (0.1 mg/kg iv) (P <: 0.01, ANOVA). Conclusions: Our investigations revealed that treatment with the 5-HT4 receptor agonlst tegaserod did not result in impairment of the mesenteric circulation in anesthetized rats. In contrast, both 5-HT3 receptor antagonists, alosetron and cilansetron, led to a retarded and sigmficant decrease in both MBF and MVC. This effect is likely to reflect a mesenteric vasoconstrictor action of the two drugs. It awaits to be determined whether this action has any bearing on the potential of 5-HT3 receptor antagonists to cause isehemic colitis in patients with IBS.

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Rotenone, Mitochondrial Electron Transport Inhibitor, Ameliorates Ischemia- Rcperfusion-lnduced Intestinal Mucosal Damage In Rats Hiroshi Ichlkawa, Tomohisa Takagi, Naoya Tomatsuri, Kazuhiko Uchiyama, Hiroshi Higashihara, Kazuhiro Katada, Yutaka Isozaki, Yuji Naito, Norimasa Yoshida, Toshikazu Yoshikawa

In ischemia/reperfnsion (I/R)-induced tissue injury, oxygen radicals can be generated by several mechanisms, including the xanthine/xanthine oxidase reaction, and the activity of NADPH oxidase and myeloperoxidase of activated leukocytes. Another potential source of oxygen radicals is thought to be mitochondrial respiration. The aim of this study was to investigate the antioxidative defense effect of mitochondrial electron transport inhibitor, rotenone, which is a naturally occurring pesticide derived from Derris and Lonchorcarpus species root and back, using the reperfusion-induced rat intestinal mucosal injury model in vivo. Intestinal ischemia was induced for 30 min by applying a small clamp to the superior mesenteric artery after ligating the celiac artery in rats. Reoxygenation was produced by removal of the clamp. Sixty minutes after reperfusion, the rats were killed by exsanguinations wa the abdominal aorta. Rotenone at a dose of 40 or 200 mg/kg body weight in 1 ml of 5% gum arabic was given to rats orally 2 hr before the vascular clamping. Both intra-luminal hemoglobin and protein levels, as indexes of mucosal damage, significantly increased from mean basal levels after 60 min of reperfusion. These increases in intra-luminal hemoglobin and protein levels were significantly inhibited by treatment with rotenone at a dose of 200 mg/kg. The concentration of thiobarbituric acid-reactive substances (TBA-RS) in the intestinal mucosa, an index of lipid peroxidation, was also significantly higher than the basal level after 60 mill of reperfusion. This increase in TBA-RS in the intestinal mucosa after I/R was inhibited by pretreatment with rotenone. Furthermore, myeloperoxidase (MPO) activity in the intestinal mucosa, an index of neutrophil infiltration, significantly increased by I/R, and pretreatment with rotenone sigmficantly reduced this increase. The content ol mucosal CINC-1 in the control groups was significantly increased compared with the levels of that in the sham-operated groups. This increase of the level of CINC- 1 was significantly inhibited by the treatment with rotenone at a dose of 200 mg/kg The results of the present study indicate that rotenone inhibited lipid peroxidation and reduced development of the intestinal mucosal inflammation induced by I/R in rats. This investigation suggests that rotenone has potential as a new therapeutic agent for reperfnsion injury.

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Involvement of Reactive Oxygen Metabolites in Gastroprotect ion Induced by lschemic Precondit ioning of the Stomach and Remote Organs Thomas Braozowski, Peter C. Konturek, Robert Paido, Agata Ptak, Stanislaw J. Konturek, Zhigniew Sliwowski, Wieslaw W. Pawfik, Eckhart G. Hahn

Ischemic preconditioning (IP) induced by repeated short ischemic episodes protects the gastric mucosa against the damage reduced by prolonged ischemia/reperfusion (I/R) but the role of reactive oxygen species (ROS) in this phenomenon has been little studied. We determined whether pretreatment with antioxidants (vitamin C, melatonin) and ROS seavang- ers (sodium dismutase (SOD), catalase) could influence the protective effect of IF of the stomach and of extragastrointestinal organs such as heart and liver against lesions evoked by I/R. The IP was induced in rats by short episodes of gastric ischemia (occlusion of celiac artery 1-5 times, 5 min each) applied 30 min prior to 3 h of I/R and compared with that achieved with IP induced by clamping of left coronary artery and hepatic artery followed 30 rain later by 3h of gastric I/R. The area of gastric lesions was determined by planimetry, gastric blood flow (GBF) measured by H2-gas clearance, the mucosal ROS and MDA content as an index of lipid peroxidation were determined and the release and expression of IL-113 and TNFa mRNA were analyzed by RT-PCR. I/R produced numerous gastric lesions and significant fall in the GBF while producing the significant rise in chemiluminescence (CI_), MDA content and plasma cytokine levels by 2-3 bids followed by overexpression of IL-1 and TNFa mRNA. Short iscbemic episodes (2x5 min) of the stomach, heart and liver by themselves failed to influence gastric lesions but significantly attenuated those produced by 3 h of I/R and this was accompanied by an increase in the GBF and sigraficant decrease in CL, MDA content and mRNA for IL-113 and TNFcc These protective and hyperemic effects of gastric, cardiac and hepatic IP against I/R damage as well as decrease in CL and plasma IL-113 and TNFa concentrations were abolished by pretreatment with vitamin C (60 rag/ kg i.g.) and melatonin (1.2 mg/kg i.g.) and by ROS scavengers, SOD (2 000 U/kg i.p.) and catalase (5 mg/kg i.p.) which by themselves failed to influence significantly the I/R injury. We conclude that: 1) gastric IP and that of remote organs, attenuate gastric lesions induced by severe I/R via suppression of lipid peroxidation and expression and subsequent release of proinflammatory cytokines IL-ll3 and TNFa; and 2) antioxidizmg agents and ROS seavangers counteract protective effect of IP against I/R lesions indicating that ROS are essential signaling

molecules that trigger gastroprotection induced by IP of the stomach and remote organs such as heart and liver.

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Effect of Buthionine Sulfoxtmine (BSO) on Redox State of Cellular and Extracellular Glutathione (GSH) and Cysteine (Cys) in HT-29 Cells Corinna L. Anderson, Thomas R. Ziegler, Dean P. Jones

Intestinal inflammation is associated with depletion of epithelial cell GSH concentrations. Metabolic interrelationships and thiol-disulfide exchange reactions between GSH/GSSG and Cys/Cystine(CySS) affect thiol-disulfide redox balance and may provide a control mechanism for cellular and/or extracellular redox in gut epithelia. This study examined the effect of BSO-induced inhibition of GSH synthesis on thlol-disul.fide redox states of cellular and extracellular GSH/GSSG and Cys/CySS. Human colon carcinoma (HT29) cells were treated with O, 1, 10 or 100 p,M BSO, then analyzed for GSH, GSSG, Cys and CySS content at 0, 4, 24 h. For extracellular studies, cells were BSO-treated for 24 h, then switched to Cys- free, CySS-supplemented medium with BSO~ Medium was analyzed for GSH, GSSG, Cys, CySS and CySSG at 0, 1, 4, 12 h. Redox state (Eh) for thlol-disulfida pairs was calculated using the Nemst equation. Treatment with 100 DM BSO for 24 h depleted 84% of cellular GSH and oxidized EhGSH/GSSG by 40 mV vs. controls (-255 mV_+ 5 mV). Despite oxidation of GSH/GSSG, cellular Cys/CySS was not significantly affected, maintaining avg Eh of -130 inV. Medium of BSO-treated cells had 50% less GSH, GSSG and CySSG than controls at 12 h, but 50 % more Cys. Medium CySS was unaffected by BSO but decreased over time. Despite these shifts, extracellular Eh GSH/GSSG and Eh Cys/CySS did not differ significantly between treated and control cells', rather, all cells re-established Eh Cys/CySS to values measured prior to medium change (-78 mV). These data show that inhibition of GSH synthesis causes marked oxidation of cellular Eh GSH/GSSG with no significant disruption of cellular or extracellular Eh Cys/CySS. This suggests that GSH is not the primary reductant for Cys/CySS, and that separate mechanisms control redox states of the two pools. Supported by NIH Grants E509047, DK55850 and Emory University Graduate Division of Biological and Biomedical Sciences.

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Quantification of Iron-Induced Lipid Peroxidation and Antioxidant Defense Mechanisms in the Human Small Intestine Using a Newly Developed Perfusion Model Freddy J. Troost, Wim H. Saris, Robert-Jan M. Brummer

Introduction Iron catalyzes formation of the highly reactive hydroxyl radical by its participa- tion in Fenton Chemistry. It is not known whether iron in chnically used dosages causes oxidative stress in the intestine in vivo in adults. To date, little is known about the antioxidant defense mechanisms against free radical attack in the small intestine. Aims and Methods This study aimed to quantify iron-induced oxidative stress and the antioxidam response following a single clinical dosage of ferrous sulfate in the human small intestine in vivo. A double lumen perfusion tube was positioned orogastrically into the small intestine in six healthy volunteers (25 +/-5 y) using normal peristalsis. The infusion tube was placed 5 cm distally from the pylorus. After positioning of the tube, a saline solution was perfused directly into the duodenum during 195 minutes at a rate of 10 ml/min. Subsequently, an iron solution containing 80 rag iron as ferrous sulphate was perfused over 30 min at 10 ml/min. Finally, saline was perfused during 60 min. From the sample port, 40 cm distal from the infusion port, intestinal fluid samples were collected at 15-min intervals. Results Malondialdehyde concentration, an indicator of total lipid peroxidation, increased significantly from 0.07 (0-0.33) p,M during saline perfusion to 3.35 (1.19-7.27) ~M at 30 rain after the start of the iron perfusion. The non-protein trolox equivalent antioxidant capacity (TEAC) increased sigraficantly from 474 (162-748) ~M during the saline perfusion to 1314 (674o1542) p,M at 30 rain after the start of the iron perfusion. Deproteination of the samples did not affect total antioxidative capacity, nor did removal of epithelial cells present in the fluid samples. Total alkaline phosphatase, an indicator of cell lysis in the small intestine during the perfusion expenment, did not change over time. The rise in TEAC was not induced by glntathione, uric acid or ascorhic acid. Conclusion Iron supplements in a clinically used dosage induce oxidative stress in the small intestine in vivo in healthy volunteers. We showed that, applying a novel intestinal perfusinn technique, an antioxidative agent of yet unknown origin is released into the gut lumen as a result of iron-induced oxidative stress. The antioxidant capacity was unexpectedly high, especially in the view of the severe dilution of the samples with saline. The antioxidant capacity was not caused by cell shedding or cell lysis, and was of non-protein origin.

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Role of Oxidants as Initiators of Ethanol Preconditioning Preventing Postischemic Leukocyte Adhesion in Murine Small Intestine Taiji Yamaguchi, Kaznhiro Kamada, Catherine Dayton, Toshikazu Yoshikawa, Rorlald J. Korthuis

[Background] Ethanol ingestion at doses equivalent to consumption of 1-2 alcoholic beverages 24 hours prior to ischemia and reperfusion (I/R) prevents postiscbemic inflammatory responses, a phenomenon referred to as late ethanol preconditioning (EPC). This study aimed to determine whether oxidants act as initiators of late EPC. [Methods] On Day 1, ethanol was administered as a bolus to C57BL/6 mice by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after administration and returned to control levels 60 rain after ingestion. 24 hrs later (on Day 2), the superior mesenteric artery was occluded for 45 rain followed by 60 min of reperfusion. The numbers of fluoreseently- labeled rolling and adherent leukocytes were counted in postcapillary venules of the small intestine in sham animals (no EPC, no l/R), in mice subjected to I/R alone or EPC+I/R, and in animals treated with Mn-TBAP (a cell-permeant superoxide dismutase mimetic),

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