Recovery of ischaemic injured porcine ileum: evidence for a contributory role of COX-1 and COX-2

BACKGROUND: We have previously shown that the non-selective cyclooxygenase (COX) inhibitor indomethacin retards recovery of intestinal barrier function in ischaemic injured porcine ileum. However, the relative role of COX-1 and COX-2 elaborated prostaglandins in this process is unclear. AIMS: To assess the role of COX-1 and COX-2 elaborated prostaglandins in the recovery of intestinal barrier function by evaluating the effects of selective COX-1 and COX-2 inhibitors on mucosal recovery and eicosanoid production. METHODS: Porcine ileal mucosa subjected to 45 minutes of ischaemia was mounted in Ussing chambers, and transepithelial electrical resistance was used as an indicator of mucosal recovery. Prostaglandins E1 and E2 (PGE) and 6-keto-PGF1alpha (the stable metabolite of prostaglandin I2 (PGI2)) were measured using ELISA. Thromboxane B2 (TXB2, the stable metabolite of TXA2) was measured as a likely indicator of COX-1 activity. RESULTS: Ischaemic injured tissues recovered to control levels of resistance within three hours whereas tissues treated with indomethacin (5x10(-6) M) failed to fully recover, associated with inhibition of eicosanoid production. Injured tissues treated with the selective COX-1 inhibitor SC-560 (5x10(-6) M) or the COX-2 inhibitor NS-398 (5x10(-6) M) recovered to control levels of resistance within three hours, associated with significant elevations of PGE and 6-keto-PGF1alpha compared with untreated tissues. However, SC-560 significantly inhibited TXB2 production whereas NS-398 had no effect on this eicosanoid, indicating differential actions of these inhibitors related to their COX selectivity. CONCLUSIONS: The results suggest that recovery of resistance is triggered by PGE and PGI2, which may be elaborated by either COX-1 or COX-2.     

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 +/- 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 +/- 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E(2) and 6-keto-PGF(1alpha) (+250 +/- 85% and +259 +/- 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE(2) and 6-keto-PGF(1alpha). The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE(2) or 6-keto-PGF(1alpha) levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE(2) and/or PGI(2) as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.     

Survivin: a novel target for indomethacin-induced gastric injury

BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal erosions and ulcers. Apoptosis is one of the mechanisms. The role of survivin, an antiapoptosis protein, in NSAID-induced gastric injury is unknown. We examined the role of survivin in NSAID-induced gastric mucosal and gastric cell injury. METHODS: We examined: (1) the effects of indomethacin (nonselective NSAID), celecoxib and NS-398 (cyclooxygenase [COX]-2-selective NSAIDs), SC-560 (a COX-1-selective NSAID), and SC-560 plus celecoxib on survivin expression and extent of injury in rat gastric mucosa; (2) the effects of indomethacin, NS-398, SC-560, and SC-560 plus NS-398 on survivin expression and injury in gastric epithelial (RGM-1) cells; and (3) the effects of survivin suppression with small interfering RNA (siRNA) on RGM-1 cell integrity at baseline and following indomethacin injury. RESULTS: Indomethacin treatment dose-dependently reduced survivin protein levels and caused severe injury of gastric mucosa and RGM-1 cells. Suppression of survivin expression with siRNA in RGM-1 cells caused cell damage and increased susceptibility to injury by indomethacin. Celecoxib treatment caused exfoliation of the mucosal surface epithelium, but neither caused deep erosions or altered survivin expression. Neither NS-398 nor SC-560 treatment altered survivin levels or produced injury in vivo or in vitro. COX-1 and COX-2 inhibitor combination caused injury in vivo and in vitro but did not decrease survivin expression. CONCLUSIONS: (1) Indomethacin, but not selective COX-1 or COX-2 inhibitors alone or in combination, reduces survivin expression in gastric mucosal cells and (2) significant reduction of survivin precedes greater severity of gastric injury.     

Modifications produced by selective inhibitors of cyclooxygenase and ultra low dose aspirin on platelet activity in portal hypertension

AIM To study the mechanism involved in the potentially beneficial effect of ultra low dose aspirin (ULDA) in prehepatic portal hypertension, rats were pretreated with selective COX 1 or 2 inhibitors (SC-560 or NS-398 respectively), and subsequently injected with ULDA or placebo.METHODS Portal hypertension was induced by portal vein ligation. Platelet activity was investigated with an in-vivo model of laser induced thrombus production in mesenteric circulation and induced hemorrhagic time (IHT). Platelet aggregation induced by ADP and dosing of prostanoid products 6-keto-PGF1α, TXB2, PGE2 and LTB4 were also performed.RESULTS The portal hypertensive group receiving a placebo showed a decreased in vivo platelet activity with prolonged IHT, an effect that was normalized by ULDA. SC-560 induced a mild antithrombotic effect in the normal rats, and an unmodified effect of ULDA. NS-398 had a mild prothrombotic action in portal hypertensive rats, similar to ULDA, but inhibited a further effect when ULDA was added. An increased 6-keto-PGF1α was observed in portal hypertensive group that was normalised after ULDA administration. TXA2 level after ULDA, remained unchanged.CONCLUSION These results suggest that the effect of ULDA on platelet activity in portal hypertensive rats,could act through a COX 2 pathway more than the COX 1,predominant for aspirin at higher doses.     

COX-2: an in vivo evidence of its participation in heat stress-induced myocardial preconditioning

OBJECTIVE: Heat stress (HS) is known to induce delayed protection against myocardial infarction. We have previously shown that inducible nitric oxide synthase (iNOS), was involved in mediating this form of preconditioning. Since iNOS and cyclooxygenase-2 (COX-2) are co-induced in various cell types, the goal of this study was to investigate whether COX-2 could also participate to the HS-induced cardioprotection. METHODS AND RESULTS: A total of 78 male Wistar rats, subjected to either heat stress (42 degrees C for 15 min) or sham anaesthesia were used for this study. Twenty-four hours later, they were treated or not with a selective COX-2 inhibitor, either celecoxib (3 mg kg(-1), i.p.) or NS-398 (5 mg kg(-1), i.p.), 30 min before being subjected to a 30-min occlusion of the left coronary artery followed by a 120-min reperfusion, in vivo. HS resulted in a marked increase in myocardial COX-2 protein expression at 24 h, associated with a significant protection against infarction (46.0+/-1.4% in sham vs. 26.8+/-3.8% in HS group) (P相似文献   

Effects of Lipopolysaccharide on Gastric Stasis: Role of Cyclooxygenase

This study was done to examine the role of cyclooxygenase (COX) in lipopolysaccharide (LPS)-induced gastroprotection and gastric stasis. In conscious rats, LPS dose and time dependently increased gastric luminal fluid accumulation. LPS decreased blood flow (laser Doppler) and prevented gastric injury from acidified ethanol at time points before significant fluid accumulation occurred. LPS increased COX-2 but not COX-1 expression. In contrast, LPS decreased gastric mucosal prostaglandin synthesis. LPS-induced gastric luminal fluid accumulation was negated by both nonselective COX inhibition with salicylate and selective COX-2 inhibition with NS-398 but not by selective COX-1 inhibition with SC-560. Neither salicylate nor NS-398 blocked LPS-induced gastroprotection. LPS-induced gastroprotection does not depend entirely on accumulation of luminal fluid and is independent of COX-1 and COX-2. However, the ability of LPS to cause gastric stasis and increase gastric luminal fluid accumulation involves COX-2. This work was supported by NIGMS Grants GM-38529 and GM-08792.     

COX-1 and COX-2 conversely promote and suppress ischemia-reperfusion gastric injury in mice

OBJECTIVE: Neutrophil activation followed by free radical production is a feature that is common to the various forms of gastric injury. However, the roles of cyclooxygenase (COX)-1 and -2 in neutrophil activation have yet to be clarified in the gastric mucosa. We examined the roles of both COX-1 and COX-2 in neutrophil activation and free radical production in ischemia-reperfusion (IR) injury in the gastric mucosa of mice. MATERIAL AND METHODS: Ischemia was induced by clamping the celiac artery for 30 min, then removing the clamp for 90 min. SC-560, a selective COX-1 inhibitor; NS-398, a selective COX-2 inhibitor; or rebamipide, a mucoprotective agent, was administered to mice 60 min before ischemia. Gastric damage was evaluated histologically and by measuring myeloperoxidase (MPO) activity. Expressions of COX protein and intercellular adhesion molecule (ICAM)-1 were evaluated by Western blot analysis and ELISA, respectively. Effects of these drugs on thiobarbituric acid reactive substances (TBARS) and gastric blood flow were also evaluated. RESULTS: COX-2 expression was induced in gastric mucosa 60 min after reperfusion, whereas COX-1 expression remained unaltered. Localization of COX-1 and ICAM-1 in IR-injured mucosa was observed mainly in endothelial cells, while COX-2 expression was detected in mesenchymal cells such as mononuclear cells, spindle-like cells and endothelial cells. SC-560 significantly decreased gastric blood flow at the reperfusion point and reduced gastric mucosal injury in IR mice. Furthermore, SC-560 pretreatment significantly reduced MPO activity, TBARS levels and ICAM-1 expression. In contrast, NS-398 significantly increased ICAM-1 expression, MPO activity and TBARS levels, and aggravated gastric damage in IR mice. Rebamipide pretreatment reduced both COX-2 expression and IR injury. CONCLUSIONS: In IR mice, COX-2 protects the gastric mucosa by down-regulating ICAM-1 expression, whereas COX-1 is involved in up-regulating reperfusion flow, thereby aggravating the mucosa.     

Functional mechanism underlying cyclooxygenase-2 expression in rat small intestine following administration of indomethacin: relation to intestinal hypermotility

BACKGROUND AND AIM: We recently reported that cyclooxygenase (COX)-2 is upregulated in the rat small intestine after administration of indomethacin, and this may be the key to non-steroidal anti-inflammatory drug (NSAID)-induced intestinal damage. The present study investigated the mechanism for COX-2 expression induced in the rat small intestine by indomethacin, in relation with ulcerogenic processes. METHODS: Animals were given indomethacin or SC-560 p.o., and the intestinal mucosa was examined 24 h later. RESULTS: Indomethacin caused hemorrhagic lesions in the small intestine, accompanied with an increase in intestinal motility, bacterial invasion and inducible nitric oxide synthase (iNOS) activity, as well as the expression of COX-2 mRNA in the mucosa. Although SC-560 did not cause any damage, this agent caused intestinal hypermotility, the bacterial invasion and the upregulation of COX-2 expression. The mucosal PGE2 content was decreased by SC-560 at 3 h but recovered 12 h later, and this recovery of PGE2 was attenuated by both atropine and ampicillin, in addition to rofecoxib. The intestinal hypermotility response to indomethacin was prevented by both 16,16-dimethyl PGE2 and atropine, but not ampicillin. Yet all these agents inhibited not only the bacterial invasion but also the expression of COX-2 and iNOS activity in the intestinal mucosa following indomethacin treatment, resulting in the prevention of intestinal lesions. CONCLUSION: These results suggest that COX-2 expression in the intestinal mucosa following the administration of indomethacin is associated with intestinal hypermotility and bacterial invasion. The intestinal hypermotility caused by COX-1 inhibition may be a key to COX-2 expression after administration of NSAIDs and their intestinal ulcerogenic properties.     

Cyclooxygenase 2 and intermittent hypoxia-induced spatial deficits in the rat

Intermittent hypoxia (IH) during sleep, a critical feature of sleep apnea, induces significant neurobehavioral deficits in the rat. Cyclooxygenase (COX)-2 is induced during stressful conditions such as cerebral ischemia and could play an important role in IH-induced learning deficits. We therefore examined COX-1 and COX-2 genes and COX-2 protein expression and activity (prostaglandin E2 [PGE2] tissue concentration) in cortical regions of rat brain after exposure to either IH (10% O2 alternating with 21% O2 every 90 seconds) or sustained hypoxia (10% O2). In addition, the effect of selective COX-2 inhibition with NS-398 on IH-induced neurobehavioral deficits was assessed. IH was associated with increased COX-2 protein and gene expression from Day 1 to Day 14 of exposure. No changes were found in COX-1 gene expression after exposure to hypoxia. IH-induced COX-2 upregulation was associated with increased PGE2 tissue levels, neuronal apoptosis, and neurobehavioral deficits. Administration of NS-398 abolished IH-induced apoptosis and PGE2 increases without modifying COX-2 mRNA expression. Furthermore, NS-398 treatment attenuated IH-induced deficits in the acquisition and retention of a spatial task in the water maze. We conclude that IH induces upregulation and activation of COX-2 in rat cortex and that COX-2 may play a role in IH-mediated neurobehavioral deficits.     

环氧合酶-2抑制剂对结肠癌细胞株的体外研究

目的 观察选择性环氧合酶-2（COX-2）抑制剂对COX-2高表达的结肠癌细胞株HT-29增殖和凋亡的影响，明确以COX2为靶点治疗结肠癌的作用途径以及与COX-2活性、表达水平的相关关系。方法 将选择性COX-2抑制剂NS-398作用于结肠癌细胞系HT29，运用MTT法检测细胞增殖状态。流式细胞仪观察NS-398对细胞凋亡的影响。进一步用逆转录聚合酶链式反应（RT-PCR）检测药物作用前后HT-29中COX-2mRNA表达。ELISA法测定前列腺素E2（PGE2）水平。Western blot检测药物作用前后细胞周期素D1、Bcl-2的表达。结果 结肠癌细胞系HT-29中COX-2 mRNA高表达，NS-398呈时间和剂量依赖性抑制HT-29细胞增殖，促进其凋亡。加入NS-398的HT-29细胞中COX-2mRNA表达水平无明显变化（P〉0．05），PGE2却显著下降（P〈0．01）。72h时空白组与NS-398（75μmol／L）处理组细胞周期素D1、Bcl-2表达水平比值分别为2．21和3．25（P〈0．01），两者表达水平随作用时间延长而下降。结论 选择性COX-2抑制剂NS-398不影响结肠癌细胞COX-2 mRNA表达水平，而与其活性相关（PGE2水平）．可能通过细胞周期素D1、Bcl-2影响结肠癌细胞系HT-29的增殖与凋亡，揭示了COX-2为靶点治疗结肠癌的分子机制。     

Participation of prostacyclin in endothelial dysfunction induced by aldosterone in normotensive and hypertensive rats

The aim of the present study was to analyze the possible involvement of vasoconstrictors prostanoids on the reduced endothelium-dependent relaxations produced by chronic administration of aldosterone in Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). For this purpose, acetylcholine (ACh) relaxations in aortic segments from both strains were analyzed in absence and presence of the cyclooxygenase-1 (COX-1) and COX-2 inhibitor indomethacin, the specific COX-2 inhibitor NS-398, the TP receptor antagonist (SQ 29 548), the thromboxane A2 (TXA2) synthase inhibitor furegrelate, and the prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP). In addition, COX-2 protein expression was studied by Western blot analysis. Release of prostaglandin E2 (PGE2) and the metabolites of PGF2alpha, TXA2, and PGI2, 13,14-dihydro-15-keto PGF2a, TXB2, and 6-keto-PGF1alpha, respectively, were measured. Treatment with aldosterone did not modify blood pressure levels in any strain. However, aldosterone markedly reduced (P<0.05) ACh-induced relaxations in segments from both strains in a similar extent. Indomethacin, NS-398, SQ 29 548, and TCP enhanced (P<0.05) ACh relaxations in both strains treated with aldosterone. Aortic COX-2 protein expression was higher in both strains of rats treated with aldosterone. In normotensive animals, aldosterone increases the ACh-stimulated aortic production of 13,14-dihydro-15-keto PGF2a, PGE2, and 6-keto-PGF1alpha (P<0.05). In SHR, ACh only increased the 6-keto-PGF1alpha production (P<0.05). It could be concluded that chronic treatment with aldosterone was able to produce endothelial dysfunction through COX-2 activation in normotensive and hypertensive conditions. PGI2 seems to be the main factor accounting for endothelial dysfunction in hypertensive rats, whereas other prostanoids besides PGI2 appear to be involved in endothelial dysfunction under normotensive conditions.     

Cardioprotection during the final stage of the late phase of ischemic preconditioning is mediated by neuronal NO synthase in concert with cyclooxygenase-2

The infarct-sparing effect of the late phase of ischemic preconditioning (late PC) lasts for 72 hours. Upregulation of both cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) has been shown to be essential to the protection in the initial stage of late PC (24 hours after PC); however, the mechanisms underlying the protection in the final stage of late PC (48 to 72 hours after PC) are unknown. Conscious rabbits were preconditioned with six cycles of 4-minute coronary occlusion/4-minute reperfusion. At 72 hours after PC, powerful protection against infarction was associated with increased myocardial levels of COX-2 mRNA, protein, and cardioprotective prostaglandins (PGI2 and PGE2). The COX-2-selective inhibitor NS-398 completely blocked the protection. Surprisingly, iNOS expression was not increased at 72 hours; instead, upregulation of neuronal NO synthase (nNOS) was evident at both the mRNA (+266+/-20%, P<0.005) and the protein levels (+195+/-66%, P<0.005), which was accompanied by an increase in myocardial nitrite/nitrate (+20+/-4%, P<0.05). The nNOS-selective inhibitors N-propyl-l-arginine or S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea completely blocked the protection of late PC at 72 hours, whereas the iNOS-selective inhibitor S-methylisothiourea had no effect. In line with these findings, the disappearance of protection at 120 hours after PC was associated with the return of nNOS mRNA, protein, and activity to control levels. Although expression of COX-2 protein was still elevated at 120 hours, only a marginal increase in PGI2 and PGE2 levels was detected. In contrast to 72 hour after PC, nNOS was not upregulated at 24 hour after PC. We conclude that (1) the cardioprotection observed in the final stage of late PC (72 hour) is mediated by nNOS, not by iNOS, in concert with COX-2, and (2) nNOS-derived NO is required to drive COX-2 activity. These data identify, for the first time, a cardioprotective role of nNOS and demonstrate, surprisingly, that the mechanism of late PC differs at 72 hours (nNOS) versus 24 hours (iNOS).     

Effects of Cyclooxygenase-2 Selective and Nitric Oxide-Releasing Nonsteroidal Antiinflammatory Drugs on Mucosal Ulcerogenic and Healing Responses of the Stomach

Effects of selective cyclooxygenase-2 (COX-2)inhibitors (NS-398) and nitric oxide (NO)-releasingaspirin (NO-ASA) on gastric ulcerogenic and healingresponses were examined in comparison with nonselective COX inhibitors such as indomethacin and aspirin(ASA). Hypothermic stress (28-30°C, 4 hr) inducedgastric lesions in anesthetized rats with an increase ofacid secretion. The lesions induced by hypothermic stress were markedly worsened by subcutaneousadministration of both indomethacin and ASA but were notaffected by either NS-398 or NO-ASA, although theincreased acid secretion during hypothermia was not affected by any of the drugs. On the otherhand, the healing of gastric ulcers induced in mice bythermal cauterization (70°C, 15 sec) wassignificantly delayed by daily subcutaneousadministration of indomethacin and ASA as well as NS-398, but not by NO-ASA.COX-2 mRNA was not detected in the intact mucosa but waspositively expressed in the ulcerated mucosa, mostpotently on day 3 after ulceration. Prostaglandin contents in the intact mouse stomach werereduced by indomethacin, ASA, and NO-ASA, while theincreased prostaglandin generation in the ulceratedmucosa was inhibited by all drugs including NS-398.After subcutaneous administration of NO-ASA topylorus-ligated rats and mice, high amounts ofNO_x were detected in both the gastriccontents and serum. In addition, both NS-398 and NO-ASAshowed an equipotent antiinflammatory effect againstcarrageenan-induced paw edema in rats as compared withindomethacin and ASA. These results suggest that bothindomethacin and ASA not only increased the mucosalulcerogenic response to stress but impaired the healingresponse of gastric ulcers as well. The former actionwas due to inhibition of COX-1, while the latter effectwas accounted for by inhibition of COX-2 and was mimicked by the COX-2-selective inhibitorNS-398. NO-ASA, although it inhibited both COX-1 andCOX-2 activity, had no deleterious effects on gastriculcerogenic and healing responses.     

A selective cyclo-oxygenase-2 inhibitor, NS-398, may improve portal hypertension without inducing gastric mucosal injury

BACKGROUND AND AIMS: Prostacyclin has been shown to play a role in hyperdynamic circulation in portal hypertension. Recently, a new subtype of cyclo-oxygenase (COX), COX-2, which acts as an inducible synthase in response to various stimuli. The aim of this study was to investigate whether COX-2 contributes to portal hypertension and whether a COX-2 blockade induces the same sort of gastric mucosal injury as a COX-1 blockade. METHODS: Portal hypertension (PHT) in rats was induced by a two-step ligation of the portal vein. The mean arterial pressure (MAP), portal pressure (PP), visceral blood flow volume (BFV), serum levels of 6-keto-prostaglandin F1alpha (PGF1alpha), thromboxane B2 (TXB2) and gastric mucosal injury induced by pure ethanol were all measured in PHT rats receiving different inhibitors (indomethacin, a highly selective COX-1 inhibitor; NS-398, a highly selective COX-2 inhibitor). Control rats treated by a sham operation were also studied. RESULTS: The NS-398 administration significantly decreased PP to the same extent as indomethacin at doses of 5 and 10 mg/kg in PHT rats after a 60 min administration, while neither inhibitor affected the control rats. Both inhibitors significantly increased PP after a 30 min administration in the PHT and control rats at a dose of 5 mg/kg while both inhibitors significantly decreased PP after 60 min administration only in the PHT rats. Portal vein ligation treatment induced a significant increase in PP and BFV of the portal vein, gastric mucosa, oesophageal mucosa and the serum levels of 6-keto-PGF1alpha and TXB2, while portal vein ligation treatment induced a significant decrease in BFV of the liver. Both blockades increased MAP and decreased PP and BFV in the splanchnic area and decreased the serum level of 6-keto-PGF1alpha and TXB2 in the PHT rats, while neither blockade modified any parameters in the control rats, except that indomethacin administration significantly decreased the BFV of the gastric mucosa. Indomethacin administration significantly increased the ulcer index (UI). The NS-398 had no effect on UI in either the PHT or control rats. Only indomethacin significantly increased the number of rats demonstrating gastric mucosal long lesions (> 2 cm) in the PHT rats. CONCLUSION: In the PHT rats, prostaglandin seemed to contribute to portal hypertension. Both COX blockades reduced PP and BFV of the portal vein and gastric mucosa. NS-398, a selective COX-2 inhibitor, may, therefore, improve portal hypertension without inducing gastric mucosal injury.     

Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism

Although the inducible isoform of NO synthase (iNOS) mediates late preconditioning (PC), it is unknown whether iNOS gene transfer can replicate the cardioprotective effects of late PC, and the role of this protein in myocardial ischemia is controversial. Thus, the cDNA for human iNOS was cloned behind the Rous sarcoma virus (RSV) promoter to create adenovirus (Ad) 5/iNOS lacking E1, E2a, and E3 regions. Intramyocardial injection of Ad5/iNOS in mice increased local iNOS protein expression and activity and markedly reduced infarct size. The infarct-sparing effects of Ad5/iNOS were at least as powerful as those of ischemic PC. The increased iNOS expression was associated with increased cyclooxygenase-2 (COX-2) protein expression and prostanoid levels. Pretreatment with the COX-2-selective inhibitor NS-398 completely abrogated the infarct-sparing actions of Ad5/iNOS, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection. We conclude that gene transfer of iNOS (an enzyme commonly thought to be detrimental) affords powerful cardioprotection the magnitude of which is equivalent to that of late PC. This is the first report that upregulation of iNOS, in itself, is sufficient to reduce infarct size. The results provide proof-of-principle for gene therapy against ischemia/reperfusion injury, which increases local myocardial NO synthase levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. The data also reveal the existence of a close coupling between iNOS and COX-2, whereby induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS. We propose that iNOS and COX-2 form a stress-responsive functional module that mitigates ischemia/reperfusion injury.     

NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2

BACKGROUND & AIMS: Selective cyclooxygenase (COX)-2 inhibitors produce less gastric damage than conventional nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that NSAIDs cause damage by inhibiting COX-1. We tested this hypothesis in rats by using a selective COX-1 inhibitor (SC-560). METHODS: The effects of SC-560, celecoxib (selective COX-2 inhibitor), or a combination of both inhibitors on gastric damage and prostaglandin synthesis were determined. Selectivity of the drugs for COX-1 vs. COX-2 was assessed in the carrageenan-airpouch model. A COX-1-preferential inhibitor, ketorolac, was also evaluated. The effects of these inhibitors on leukocyte adherence to vascular endothelium and on gastric blood flow were assessed. RESULTS: SC-560 markedly reduced gastric prostaglandin synthesis and platelet COX-1 activity, but spared COX-2 and did not cause gastric damage. Celecoxib did not affect gastric prostaglandin E(2) synthesis and did not cause gastric damage. However, the combination of SC-560 and celecoxib invariably caused hemorrhagic erosion formation, comparable to that seen with indomethacin. Ketorolac caused damage only at doses that inhibited both COX isoforms, or when given with a COX-2 inhibitor. Celecoxib, but not SC-560, significantly increased leukocyte adherence, whereas SC-560, but not celecoxib, reduced gastric blood flow. CONCLUSIONS: Inhibition of both COX-1 and COX-2 is required for NSAID-induced gastric injury in the rat.     

Aggravation by Selective COX-1 and COX-2 Inhibitors of Dextran Sulfate Sodium (DSS)-Induced Colon Lesions in Rats

We examined the effect of cyclooxygenase (COX) inhibitors on dextran sulfate sodium (DSS)-induced ulcerative colitis in rats and investigated the role of COX isozymes in the pathogenesis of this model. Experimental colitis was induced by treatment with 2.5% DSS in drinking water for 6 days. Indomethacin (a nonselective COX inhibitor), SC-560 (a selective COX-1 inhibitor), or celecoxib (a selective COX-2 inhibitor) was given PO twice daily for 6 days, during the first 3 or last 3 days of the experimental period. Daily treatment with 2.5% DSS for 6 days caused damage to the colon, with a decrease in body weight gain and colon length as well as an increase of myeloperoxidase (MPO) activity. All COX inhibitors given for 6 days significantly worsened the severity of DSS-induced colonic damage with increased MPO activity. The aggravation was also observed by SC-560 given for the first 3 days or by celecoxib given for the last 3 days. The expression of COX-2 mRNA in the colon was upregulated on day 3 during DSS treatment, with significant increase of prostaglandin E₂ PGE₂ production. The PGE₂ content on day 3 during DSS treatment was inhibited by both indomethacin and SC-560, but not by celecoxib; on day 6 it was suppressed by both indomethacin and celecoxib, but not SC-560. These results suggest that endogenous prostaglandins (PGs) afford protection against colonic ulceration, yet the COX isozyme responsible for the production of PGs differs depending on the stage of ulceration; COX-1 in the early stage and COX-2 in the late stage.     

Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning

Cyclooxygenase-2 (COX-2) is known to mediate the cardioprotective effects of the late phase of ischemic preconditioning (PC); however, the signaling pathways involved in COX-2 induction following ischemic PC are unknown. In addition, although inducible nitric oxide synthase (iNOS) has been identified as a co-mediator of late PC together with COX-2, the interaction between iNOS and COX-2 in the heart is unknown. Using conscious rabbits, we found that the induction of COX-2 expression 24 hours after ischemic PC was blocked by pretreatment with inhibitors of protein kinase C (PKC), Src protein tyrosine kinases (PTKs), and nuclear factor-kappaB (NF-kappaB) but not by inhibitors of NOS or scavengers of reactive oxygen species (ROS). The selective iNOS inhibitors SMT and 1400W, given 24 hours after PC, abrogated the increase in myocardial prostaglandin E2 (PGE2) and 6-keto-PGF1alpha, whereas the selective soluble guanylate cyclase inhibitor ODQ had no effect. COX-2 selective inhibitors (celecoxib and NS-398) did not affect iNOS activity. These results demonstrate that (i) ischemic PC upregulates cardiac COX-2 via PKC-, Src PTK-, and NF-kappaB-dependent signaling pathways, whereas generation of NO and ROS is not necessary, and (ii) the activity of newly synthesized COX-2 following PC requires iNOS-derived NO whereas iNOS activity is independent of COX-2-derived prostanoids, indicating that COX-2 is located downstream of iNOS in the protective pathway of late PC. The data also indicate that iNOS modulates COX-2 activity via cGMP-independent mechanisms. To our knowledge, this is the first demonstration that iNOS-derived NO drives prostanoid synthesis by COX-2 in the heart. NO-mediated activation of COX-2 may be a heretofore unrecognized mechanism by which NO exerts its salubrious effects in the late phase of PC.     

Par-4, a proapoptotic gene, is regulated by NSAIDs in human colon carcinoma cells

BACKGROUND & AIMS: Many reports indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) have antineoplastic effects, but the precise molecular mechanism(s) responsible are unclear. We evaluated the effect of cyclooxygenase (COX) inhibitors (NSAIDs) on human colon carcinoma cells (HCA-7) and identified several genes that are regulated after treatment with NS-398, a selective COX-2 inhibitor. METHODS: Differential display polymerase chain reaction cloning techniques were used to identify genes regulated by treatment with NSAIDs and selective COX-2 inhibitors. RESULTS: A prostate apoptosis response 4 (Par-4) gene was up-regulated after NSAID treatment. Par-4 was first isolated from prostate carcinoma cells undergoing apoptosis, and expression of Par-4 sensitized cancer cells to apoptotic stimuli. Par-4 levels were increased in cells treated with COX inhibitors such as NS-398, nimesulide, SC-58125, and sulindac sulfide. Treatment of HCA-7 cells with these agents also induced apoptotic cell death. CONCLUSIONS: The results suggest that regulation of Par-4 contributes to the proapoptotic effects of high-dose COX inhibitors (NSAIDs) by serving as a downstream mediator leading to initiation of programmed cell death.