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Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress
J. Physiol. (Paris) 94 (2000) 83−91 © 2000 Elsevier Science Ltd. Published by Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0928425700001455/FLA
Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress Tomasz Brzozowskia, Peter Ch. Konturekb, Stanislaw J. Kontureka*, Danuta Drozdowicza, Robert Pajdoa, Michat Pawlika, Iwona Brzozowskaa, Eckhart G. Hahnb a
Department of Physiology, Jagiellonian University School of Medicine, Cracow, Poland b Department of Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany
Abstract — Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg⋅kg–1 i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg⋅kg–1 i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg⋅kg–1 i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg⋅kg–1 i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5–30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg⋅kg–1 i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS, whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 µg⋅kg–1 i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors. © 2000 Elsevier Science Ltd. Published by Éditions scientifiques et médicales Elsevier SAS adaptive cytoprotection / stress damage / mild stress / cyclooxygenase-1 / cyclooxygenase-2 / prostaglandin E2 / gastric blood flow / NS-398 / meloxicam
1. Introduction Endogenous prostaglandins (PG) play a crucial role in the maintenance of gastric mucosal integrity and contribute to gastroprotection and short or long-term adaptation of gastric mucosa to the damaging action of topical irritants [2, 19, 22, 30, 31]. Short-term adaptation known as adaptative cytoprotection was originally introduced by Robert et al. [30, 31] to describe the protective action of certain mild irritants such as 20% ethanol, 5% NaCl and 5 mM taurocholate against the damage induced by these agents applied intragastrically in larger concentrations. This action of mild irritants has been attributed to the protective action of endogenous PG [19, 31] but recently the importance of other protective mediators such as nitric oxide (NO) or sensory nerves were implicated in this phenomenon [7, 14, 32]. * Corresponding author
It is generally accepted that at least two isoforms of the principle enzyme responsible for PG synthesis cyclooxygenase (COX) exist; constitutive COX-1 that provides PG involved in physiological reactions and inducible COX-2 that can be induced by various cytokines, growth factors and endotoxins [6, 9, 36]. PG derived from COX-1 were shown to be important in the maintenance of mucosal integrity, whereas products of the COX-2 have been implicated in inflammatory reactions [6, 9]. Exogenous PG were shown to protect gastric mucosa against stress lesions [38] but it remains unknown which COX is involved in adaptive cytoprotection induced by mild stress against severe stress. Mild stress was shown to prevent gastric lesions induced by severe stress and this effect was attributed to the activity of the adrenomedullary system [12] but the possibility that PG derived from COX-2 are involved in this adaptation has not been examined. In this study, selective COX-1 and COX-2 inhibitors
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were used to determine the contribution of the products of these enzymes in gastric blood flow (GBF) and mucosal protective action of mild stress against gastric lesions induced by stronger stress. In addition, we attempted to assess the expression of COX-1 and COX-2 by RT-PCR in intact gastric mucosa and that exposed to severe stress with or without pretreatment with mild stress. 2. Material and Methods 2.1. Production of stress lesions Stress lesions were provoked by placing animals into individual Bollman cages to produce restrain and immersing them into a water bath for 3.5 h at 23 °C to the level of the xyphoid as described in details in our previous studies [1, 20]. Mild stress was also induced by water immersion and restraint but for a short time starting from 5 min up to 2 h before the subsequent exposure to a stronger stress lasting 3.5 h in order to examine which period of stress would give optimal protective effect against severe WRS. Several groups of rats were exposed to mild WRS at 23 °C followed 1 h later by 3.5 h of WRS with or without the pretreatment with: 1) vehicle (saline); 2) N-[2(cyclohexyloxy)-4-nitrofenyl]-methanesulphonamide (NS-398) (10 mg⋅kg–1 i.g.), a highly selective COX-2 inhibitor [11]; 3) resveratrol (10 mg⋅kg–1 i.g.), a selective COX-1 inhibitor; 4) meloxicam (2 mg⋅kg–1 i.g.), an agent that was shown to inhibit preferentially COX-2 over COX-1 [8, 16, 34]; and 5) indomethacin (5 mg⋅kg–1 i.p.), a non-selective inhibitor of COX. At the dose used in the present study, indomethacin has been shown previously to inhibit gastric PGE2 synthetic capacity by ∼ 90% without causing mucosal damage [21]. The dose of NS-398 was selected on the basis of previous studies showing that it blocks completely PGE2 production in the exudate of air-pouch inflammation but does not inhibit gastric PGE2 content [23]. None of the other COX inhibitors used in this study by themselves produced gastric lesions at the dose tested. Several groups of rats consisting of 6–8 animals received the following treatments: 1) vehicle followed 30 min later by exposure to standard 3.5 h of WRS; 2) mild WRS (5 min–2 h) followed 1 h later by standard WRS; 3) indomethacin (5 mg⋅kg–1 i.p.) followed 90 min later by 3.5 h WRS; 4) NS-398 (10 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 5) resveratrol (5 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 6) meloxicam (2 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 7) indomethacin (5 mg⋅kg–1 i.p.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS; 8) NS-398 (10 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and then followed 60 min
later by 3.5 h WRS; 9) resveratrol (5 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS; and finally 10) meloxicam (2 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS. NS-398 was dissolved in absolute ethanol (5 mg⋅mL–1) and was further diluted in saline containing 0.2% methylocelulose and administered in a total volume of 1 mL. Control rats received the corresponding vehicle. In another group of animals treated with COX-1 and COX-2 inhibitors before the combination of mild WRS plus severe WRS, the effect of prostaglandin replacement therapy with 16,16 dimethyl PGE2 (Upjohn, Kalamazoo, MI, USA) was determined. For this purpose, a synthetic PGE2 analogue was administered at a dose of 1 µg⋅kg–1 i.p. 15 min before treatment with each COX-1 or COX-2 inhibitor followed 30 min later by mild WRS for standard 15 min and then challenged 60 min later with 3.5 h WRS. 2.2. Determination of gastric blood flow (GBF) and mucosal generation of PGE2 To evaluate the influence of mild WRS against more severe stress without or with the pretreatment with COX-1 and COX-2 inhibitors, the rats were lightly anaesthetized with ether and the abdomen was opened by a midline incision. The stomach was exposed to assess the GBF using H2-gas clearance technique as described previously [3]. Briefly, the double electrodes of a electrolytic regional blood flowmeter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the mucosa. One of these electrodes was used for the local generation of gaseous H2 and another for the measurement of tissue H2. With this method, the H2 generated locally is carried out by the blood stream, while the polarographic current detector reads out decreasing tissue H2. The clearance curve of tissue H2 was used to calculate an absolute flow rate (mL⋅min–1⋅100 g–1) in the oxyntic gland area as described previously [2, 3]. The measurements were made in three areas of the gastric oxyntic mucosa and the mean values of these measurements were calculated and expressed as percent changes from those recorded in intact animals. For the PGE2 measurement, the biopsy samples of the oxyntic mucosa were taken (about 100 mg) immediately after the animals had been killed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [21]. Briefly, the mucosal sample was placed in preweighed Eppendorf vials and 1 mL of Tris buffer (50 mM, pH 9.6) was added to each vial. The samples were finely minced (about 15 s) with scissors, then washed and centrifuged for 10 s, the pellet being resuspended again in 1 mL Tris. Then each sample was incubated
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on a Vortex mixer (Unipan, Warsaw, Poland) for 1 min and centrifuged for 15 s. The pellet was weighed, and the supernatant was transferred to a second Eppendorf vial containing indomethacin (10 mM) and kept at –20 °C until RIA. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The capability of the mucosa to generate PGE2 was expressed in nanograms per gram of wet tissue weight. 2.3. Expression of COX-1 and COX-2 mRNA transcripts in the gastric mucosa determined by RT-PCR COX-1 and COX-2 mRNA were determined by RT-PCR in the gastric mucosa of intact rats or those exposed to severe WRS with or without the pretreatment with mild WRS applied during a standard period of 15 min. Samples of the gastric oxyntic mucosa (about 500 mg) were scraped off on ice using a glass slide and then immediately snapped frozen in liquid nitrogen, and stored at –80 °C. Total RNA was isolated from the gastric oxyntic mucosa according to Chomczynski and Sacchi [4] using a rapid guanidinum isothiocyanate/phenol chloroform single step extraction kit from Stratagene (Stratagene GmbH, Heidelberg, Germany). Following precipitation, the RNA was resuspended in RNase-free TE buffer and the concentration was estimated by absorbance at 260 nm wavelength. Samples were frozen at –80 °C until analysis. First strand cDNA was synthesized from total cellular RNA (5 µg) using 200 U Strata Script TM reverse transcriptase and oligo (dT) primers (Stratagene GmbH, Heidelberg, Germany). After the reverse transcription, the transcriptase activity was destroyed by heating, and the cDNA were stored at –20 °C until PCR. The primers for COX-1 and COX-2 were synthesized by Biometra (Göttingen, Germany). The primer sequences were designed according to the published cDNA sequence for the rat cyclooxygenases [9, 17, 28]. The COX-1 primer sequences were as follows: upstream, 5’-AGC CCC TCA TTC ACC CAT CAT TT; downstream, 5’-CAG GGA CGC CTG TTC TAC GG. The expected length of this PCR product was 561 bp. The COX-2 primer sequences were as follows: upstream, 5’-ACA ACA TTC CTT CCT TC; downstream, 5’-CCT TAT TTC CTT TCA CAC C. The expected length of this PCR product was 201 bp. Concomitantly, amplification of control rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on the same samples to verify RNA integrity. The primer sequences for GAPDH were purchased from Clontech (Heidelberg, Germany). Reaction mixtures for PCR contained cDNA templates, 50 pmol of each primer, and 2.5 U of Taq DNA
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polymerase (Serva GmbH, Heidelberg) in 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTPs in a volume of 50 µL. RT blanks (no RNA included) and PCR blanks (no cDNA products included) were included in each analysis. DNA amplification was carried out under the following conditions; denaturation at 94 °C for 1 min, annealing at 60 °C for 45 s and extension at 72 °C for 45 s. To maximize amplification specificity, Taq DNA polymerase was added to the PCR mixture during the hot start of cycle 1. The COX-1 and COX-2 genes were amplified for 30 cycles. Each PCR-product (8 µL) was electrophoresed on a 1.5% agarose gel stained with ethidium bromide, and then visualized under UV light. Location of predicted PCR product was confirmed by using a 100-base pair ladder (Gibco BRL/Life Technologies, Eggenstein, Germany) as standard marker. To avoid PCR contamination, PCR reactions were prepared in a dedicated area used only for PCR and the PCR product were opened in a laminar flow hood separated from the PCR preparation area.
3. Results 3.1. Stress-induced gastric lesions and demonstration of adaptive cytoprotection by mild stress As shown in figure 1, exposure of rats to a standard 3.5 h WRS produced numerous gastric lesions (approximately 18–22 per stomach) that were identified predominantly in the oxyntic mucosa as bleeding erosions. In contrast, mild WRS lasting from 5 to 30 min by itself failed to produce gross gastric lesions (data not shown) but significantly reduced the lesions provoked by more severe standard stress, the optimal protective effect being obtained with 15 min mild WRS (figure 1). In further studies, mild WRS lasting for 15 min was used and it caused about 80% reduction in lesions induced by standard severe stress. The gross appearance of such stomach with the lesions induced by the exposure to 3.5 h WRS with or without pretreatment with 15 min mild WRS is presented in figure 2 (lower), BAs shown in figure 2 (upper), the pretreatment with mild WRS for 15 min significantly attenuated gross mucosal lesions induced by more severe stress. Extension of the duration of mild WRS to 30 min was not more effective in reduction of lesions induced by standard stress when compared to that recorded in animals subjected to 15 min mild WRS (figure 1). Further extension of the duration of WRS up to 1 h did not result in any reduction in the number of gastric lesions caused by standard severe stress, while the application of 2 h of WRS aggravated lesions induced by the subsequent exposure to this standard severe stress (figure 1).
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Figure 1. Mean number of gastric lesions and gastric mucosal blood flow (GBF) in rats exposed to 3.5 h water immersion and restraint stress (WRS) without or with pretreatment with mild WRS ranging from 5 to 120 min. Results are mean ± SEM of 8 rats. Asterisk indicates a significant change (P < 0.05) as compared to the value obtained in rats without pretreatment with mild WRS. Cross indicates a significant change as compared to animals pretreated with mild WRS for 15 min followed by exposure to 3.5 h of more severe stress. Double cross indicates a significant change as compared to the value recorded in gastric mucosa exposed to severe WRS.
GBF in intact gastric mucosa averaged 46 ± 6 mL⋅min–1⋅100 g–1 tissue (taken as 100%) and this was reduced by about 49% in rats subjected to 3.5 h of severe stress (figure 1). Application of mild WRS before a standard 3.5 h severe stress attenuated the fall of GBF caused by this stress and the maximal increase of GBF was reached in animals pretreated with 15 min of mild WRS (figure 1). With the prolongation of mild WRS to 1 h before more severe stress, the fall in GBF was not significantly different from that recorded with standard WRS but further extension of the mild WRS up to 2 h caused a fall in GBF greater than that observed with severe stress alone (figure 1). 3.2. Effect of suppression of PGE2 generation on gastroprotection induced by mild WRS The generation of PGE2 in the intact gastric mucosa averaged 148 ± 12 ng⋅g–1 of wet tissue weight but when the mild WRS was applied alone (for 15 min), a significant increase in PGE2 generation was recorded, which reached the value of 193 ± 8 ng⋅g–1 of wet tissue weight. Such standard mild WRS, which by itself failed to produce gastric lesions, resulted in a significant increase in the GBF by about 23% as compared to that in intact gastric mucosa (data not
Figure 2. Gross appearance of the rat gastric mucosa after the exposure to 3.5 h of stress without or with pretreatment with mild WRS. In rat exposed to 3.5 h of severe stress, numerous gastric lesions are evident (lower panel), whereas in rat pretreated with standard 15 min of mild WRS before the exposure to severe stress, the number of gastric lesions is markedly attenuated (upper panel).
shown). In contrast, the exposure to 3.5 h WRS alone which caused gastric lesions, attenuated significantly the GBF and PGE2 generation as compared to those in the intact mucosa. Pretreatment with mild WRS significantly reduced gastric lesions induced by more severe stress and attenuated the decrease in GBF and PGE2 generation induced by this severe stress (figure 3). The administration of indomethacin (5 mg⋅kg–1 i.p.) 90 min before the application of 3.5 h of stress, suppressed mucosal generation of PGE2 by about 90% and increased significantly the mean number of gastric lesions, the effect being accompanied by a significant fall in the GBF (figure 3). When indomethacin was applied before the combination of mild WRS plus severe WRS, it abolished completely the reduction in gastric lesions and accompanying increase in the GBF induced by the pretreatment with mild WRS (figure 3).
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Figure 3. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with indomethacin (5 mg·kg–1 i.p.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa pretreated with mild WRS before exposure to 3.5 h severe stress.
Figure 4. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with resveratrol (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals pretreated with standard mild WRS before exposure to 3.5 h severe stress.
3.3. Effect of specific and non-specific COX-1 and COX-2 inhibitors on adaptive cytoprotection induced by mild WRS against severe stress
meloxicam, applied i.p. in a dose of 2 mg⋅kg–1, aggravated significantly gastric lesions induced by 3.5 h severe stress and decreased significantly the GBF recorded in animals exposed to this severe stress (figure 5). Meloxicam, which suppressed the PGE2 generation, abolished completely the protective and hyperemic effects of mild 15 min WRS on subsequent more severe stress (figure 5).
Figure 4 shows the effects of pretreatment with resveratrol (10 mg⋅kg–1 i.g.), a specific inhibitor of COX-1, on the gastric lesions induced by severe stress with or without pretreatment with mild WRS. Pretreatment with resveratrol, which produced small and not significant decrease in the PGE2 generation, failed to influence significantly the number of lesions caused by exposure to 3.5 h severe stress and accompanying fall in the GBF. Such pretreatment with resveratrol reversed, in part, the mild WRS-induced gastric protection and accompanying increase in the GBF and attenuated significantly the rise in the PGE2 caused by the mild WRS (figure 4). Pretreatment with NS-398, a specific inhibitor of COX-2 given in a dose of 10 mg⋅kg–1 (i.g.), failed to affect significantly the number of gastric lesions induced by severe stress (3.5 h) and accompanying fall in GBF but significantly attenuated the protection against this stress induced by 15 min mild WRS and completely reversed the rise in GBF evoked by mild WRS (figure 5). Such pretreatment with NS-398 failed to influence significantly the PGE2 generation in rats exposed to severe stress with or without the pretreatment with mild WRS (figure 5). Pretreatment with
3.4. Effect of PG replacement therapy with exogenous PGE2 on adaptive cytoprotection induced by mild WRS As shown in figure 6, the pretreatment with COX-1 and COX-2 inhibitors before the combination of mild WRS plus severe stress produced similar alterations in the number of gastric lesions and GBF as presented in figures 3, 4. Administration of PGE2 (1 µg⋅kg–1 i.p.) counteracted, in part, the inhibitory effect of nonselective COX inhibitors on the mild WRS-induced protection against severe stress and accompanying alterations in GBF (figure 6). Such pretreatment with PGE2 completely abolished the increase in the number of lesions and the accompanying fall in GBF induced by administration of the selective COX-2-inhibitor, NS-398, or selective COX-1-inhibitor, resveratrol (figure 6).
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Figure 5. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with meloxicam (2 mg·kg–1 i.p.) or NS-398 (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa of rats pretreated with mild WRS before exposure to 3.5 h severe stress.
3.5. Expression of COX-1 and COX-2 mRNA by RTPCR in gastric mucosa of rats exposed to vehicle or severe stress with or without the pretreatment with mild WRS Figure 7A–C shows the mRNA expression of β-actin, COX-1 and COX-2 in the gastric mucosa of control rats treated with vehicle or those exposed to 3.5 h stress alone, mild WRS alone or their combination. The expression of mRNA for β-actin was wellpreserved in the mucosal samples taken from rats treated with vehicle (control) or those exposed to severe stress with or without the pretreatment with mild WRS (figure 7A). The COX-1 mRNA was detectable in the vehicle-treated gastric mucosa as well as in the mucosa exposed to severe stress with or without pretreatment with mild WRS (figure 7B). In contrast, COX-2 mRNA was undetectable in vehicle control animals but has been traced in rats exposed to mild WRS alone or severe stress with or without the combination with mild WRS (figure 7C). 4. Discussion In the present study, we confirmed that the exposure of rats to mild WRS protects the stomach against
Figure 6. Mean lesion number and the GBF in the gastric mucosa of rats exposed to standard 15 min mild WRS followed by exposure to 3.5 h of severe stress without or with the pretreatment with 16,16 dimethyl PGE2 (1 µg·kg–1 i.p.) added to indomethacin (5 mg·kg–1 i.p.), meloxicam (2 mg·kg–1 i.p.), resveratrol (10 mg·kg–1 i.g.) or NS 398 (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa of rats treated with mild WRS plus 3.5 h of severe stress without pretreatment with a PGE2 analogue.
gastric mucosal lesions provoked by severe stress probably via the phenomenon of adaptive cytoprotection similar to that afforded by mild irritants [19, 30, 31]. However, this protective effect was observed only when shorter periods of mild WRS lasting from 5 to 30 min was employed before the severe stress. An extension of mild WRS up to 2 h not only failed to protect the gastric mucosa but even augmented the stress-induced gastric lesions. Furthermore, such pretreatment with 15 min of mild WRS (which attenuated lesions caused by severe stress) produced significant rise in PGE2 generation and GBF in the gastric mucosa suggesting that this enhancement in the gastric microcirculation was probably due to increased generation of mucosal PG. This is supported by the fact that pretreatment with indomethacin, a non-selective inhibitor of COX, which strongly suppressed PGE2 generation, completely abolished the protective and hyperemic effects of mild WRS against lesions provoked by severe stress. In contrast, the administration of NS-398, a specific COX-2 inhibitor, or resveratrol, a specific COX-1 inhibitor, reversed only in part the gastroprotection and accompanying rise in GBF induced by mild WRS and these effects were fully restored by the addition of exogenous PGE2 to this
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Figure 7. Analysis of reverse transcriptase polymerase chain reaction (RT-PCR)-amplified β-actin, COX-1 and COX-2 products using specific primers. M = PCR size marker (PhiX 174 RF DNA Hae III Digest, New England Biolabs, USA); lane 1, intact gastric mucosa; lane 2, 3.5 h stress alone; lane 3, standard mild WRS alone; lane 4, standard mild WRS plus 3.5 h severe stress.
COX-2 or COX-1 inhibitor. Moreover, the strong signals for COX-1 mRNA was detected in the vehicle treated control gastric mucosa as well as in the mucosa of rats exposed to mild WRS, severe stress or their combination. In contrast, COX-2 mRNA was undetectable in the intact vehicle-treated gastric mucosa but was well-defined in the mucosa of animals exposed to mild WRS or severe stress with or without pretreatment with mild WRS suggesting that the upregulation of COX-2 mRNA with excessive production of protective PG could contribute to adaptive cytoprotection induced by mild WRS. Previous studies have established that PG synthesis depends upon the activity of cyclooxygenase (COX), a rate limiting enzyme in the synthesis of eicosanoids [6, 34]. Two isoforms of COX were identified in many cells; a constitutive enzyme designated as COX-1 and inducible isoform known as COX-2 [17, 24]. Under
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physiological conditions, prostanoid synthesis depends upon the availability of arachidonic acid and the enzymatic activity of COX-1, which is a major target for NSAIDs resulting in mucosal damage in the stomach [29]. In contrast, COX-2 is not constitutively expressed in most of the tissues but could be induced by a variety of stimuli including interleukin-1, tumour necrosis factor (TNF)-α, growth factors, lipopolisacharide and oxidative stress [24, 26, 37]. Downregulation of COX-1 associated with a decrease of cytoprotective prostanoids was recently implicated in the exacerbation of liver injury by ethanol [27]. On the other hand, the upregulation of COX-2 due to ethanol treatment in that study [27] resulted in increased synthesis of inflammatory and vasoactive eicosanoids. We confirmed our previous observations [1, 20] that exposure of the rats to 3.5 h stress induced by cold and restraint produces gastric lesions and that this effect is accompanied by a decrease in PGE2 generation and a marked fall in GBF. Non-topical irritant such as mild WRS, which by itself failed to produce gastric lesions, or alter increased PGE2 generation, prevented the formation of lesions induced by severe stress, supporting previous findings with mild irritants [35] that endogenous PG are essential for the development of adaptive cytoprotection. We attempted in this study to determine the relative contribution of COX-1 and COX-2 to stress ulcerogenesis and adaptive cytoprotection induced by mild WRS. In agreement with previous studies [6, 36, 37], we found that in the normal stomach COX-1 is predominantly expressed, whereas COX-2 mRNA is not. Strong signals for COX-1 mRNA were also detected in gastric mucosa of rats exposed to mild or severe WRS and in those pretreated with mild WRS before exposure to severe stress. It is of interest that an overexpression of COX-2 mRNA was observed in the mucosa exposed to mild or severe stress applied alone or in combination, suggesting that the upregulation of the gene for COX-2 may contribute to the protective action of mild WRS. The overexpression of COX-2 mRNA during severe stress might be due to the deficit of PGE2 generation in the gastric mucosa as demonstrated in the present study. This notion is in keeping with recent finding by Davies et al. [5], who found an upregulation of COX-2 mRNA in a stomach acutely treated with aspirin applied at a dose that induced gastric damage and almost complete suppression of mucosal generation of PGE2. It was concluded [5] that this elevation in COX-2 expression was not a specific response to injury because topically applied irritants such as ethanol, indomethacin or salicylate failed to alter significantly both, COX-2 mRNA and COX-2 protein expression. In contrast to this observation, Mizuno et al. [25] showed that COX-2 mRNA and COX-2 protein levels in mice stomach were markedly
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increased after ethanol-induced acute mucosal injury and during the healing of chronic gastric ulcers. As mentioned before, the upregulation of COX-2 mRNA observed in our present study could be triggered by the depletion of endogenous PG caused by severe stress. This is supported by the observation that replacement therapy with exogenous PGE2 added to specific COX-2 inhibitor to cover the deficiency of PG, attenuated significantly the deleterious effect of this inhibitor. In another study, the increased expression of COX-2 mRNA induced by aspirin at a dose that probably suppressed mucosal generation of PGE2, was significantly diminished by the administration of exogenous PGE2 [5]. Thus, our study demonstrates that PG derived from both COX-1 and COX-2 enzymes seem to mediate the protective response to mild stressor. This is supported by the fact that non-specific inhibitor of COX, indomethacin at the dose that suppressed PGE2 generation at about 90%, completely abolished the protective and hyperemic response to mild WRS. It is of interest, that meloxicam, shown to inhibit preferentially COX-2 but also inhibiting COX1 [8], antagonized almost completely these responses in the manner similar to that observed with indomethacin. In contrast, NS-398 which failed to alter PGE2 generation in gastric mucosa significantly attenuated the protection and accompanying hyperemia induced by mild WRS, and a similar degree of reversal of the protective effect of mild WRS was obtained in animals pretreated with specific COX-1 inhibitor, resveratrol. Thus, it is reasonable to assume that this adaptive cytoprotection could be completely antagonized by agents that suppress both COX-1 and COX-2 pathways or impaired by inhibition of either, COX-1 or COX-2 pathway. This notion is in keeping with recent observations by Gretzer et al. [13] that PG generated by COX-2 are involved in adaptive cytoprotection induced by topically applied mild irritant. These authors [33] concluded that COX-2 is constitutively expressed and may play a significant role in adaptive cytoprotection induced by topically acting mild irritant. In agreement with this notion, the COX-2 mRNA and COX-2 protein were recently identified in endothelia of gastric mucosal microvessels [33]. COX-1 is generally regarded to be a constitutively expressed enzyme, whereas COX-2 is usually referred to as the inducible enzyme isoform [16]. However, COX-2 was shown to be constitutively expressed in some tissues and there is evidence of the inducibility of COX-1 in gastric mucosa of rats treated chronically with endotoxin [10]. It is of interest that immunoreactivity of COX-2 was detected in the prepyloric and fundic regions of the rat gastric mucosa suggesting that COX-2 could be expressed in unstimulated stomach [15]. We were not able to show expression of COX-2 mRNA in intact animals similarly to a recent
report [10] but an overexpression of COX-2 mRNA was observed in rats exposed to mild and severe stress or after their combination suggesting that in addition to COX-1, COX-2-derived prostaglandins play an important role in adaptive cytoprotection induced by mild WRS. This is in keeping with the finding that the induction of COX-2 was triggered by ischemiareperfusion and accompany the healing of chronic gastric ulcerations thus indicating that COX-2-derived PG are essential to ensure ulcer healing [18, 25]. On the other hand, that COX-1 was the primary source of prostaglandins responsible for this mild stress is supported by our finding that indomethacin, which is more selective for COX-1 than COX-2 [16, 34], reversed completely the protection induced by mild WRS against severe stress, whereas NS-398, a highly selective COX-2 inhibitor in the same experimental conditions, had only partial inhibitory effect on the stress lesions and failed to influence accompanying enhancement in PGE2 generation induced by mild WRS. References [1]
Brzozowski T., Konturek S.J., Majka J., Dembinski A., Drozdowicz D., Epidermal growth factor, polyamines and prostaglandins in healing of stress induced gastric lesions in rats, Dig. Dis. Sci. 38 (1993) 276–283. [2] Brzozowski T., Konturek P.Ch., Konturek S.J., Stachura J., Gastric adaptation to aspirin and stress enhances gastric mucosal resistance against the damage by strong irritants, Scand. J. Gastroenterol. 31 (1996) 118–125. [3] Brzozowski T., Konturek S.J., Sliwowski Z., Pajdo R., Drozdowicz D., Stachura J., Role of β-adrenoceptors in gastric mucosal integrity and gastroprotection induced by epidermal growth factor, Digestion 58 (1997) 319–331. [4] Chomczynski P., Sacchi N., Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. [5] Davies N.M., Sharkley K.A., Asfaha S., MacNaughton W.K., Wallace J.L., Aspirin causes rapid up-regulation of cyclooxygenase-2 expression in the stomach of rats, Alim. Pharmacol. Ther. 11 (1997) 1101–1108. [6] Eberhart C.E., Dubois R.N., Eicosanoids and the gastrointestinal tract, Gastroenterology 109 (1995) 285–301. [7] Endo K., Kao J., Bomek M.J., Leung F.W., Mechanism of gastric hyperemia induced by intragastric hypertonic saline in rats, Gastroenterology 104 (1993) 114–121. [8] Engelhardt G., Bogel R., Schmitzer Ch., Utzman R., Meloxicam; Influence on arachidonate metabolism. Part 1 in vitro findings, Biochem. Pharmacol. 51 (1996) 21–28. [9] Feng L., Xia Y., Garcia G.E., Hwang D., Wilson C.B., Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha and lipopolysaccharide, J. Clin. Invest. 95 (1995) 1669–1675. [10] Ferraz J.G., Sharkey K.A., Reuter B.K., Asfaha S., Tigley A.W., Brown M.L., McKnight W., Wallace J.L., Induction of cyclooxygenase-1 and -2 in the rat stomach during endotoxemia: role in resistance to damage, Gastroenterology 3 (1997) 195–204.
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[11] Futaki N., Yoshikawa K., Hamasaka Y., Arai I., Higuchi S., Izuka H., Otomo S., NS-398 a novel non-steroidal antiinflammatory drug with potent analgesic and antipyretic effects which causes minimal stomach lesions, Gen. Pharmacol. 24 (1993) 105–110. [12] Glavin G.B., Lockhart L.K., Rockman G.E., Hell A.M., Kierhan K.M., Evidence of cross-stressor induced adaptive gastric cytoprotection, Life Sci. 41 (1987) 2223–2227. [13] Gretzer B., Ehrlich K., Maricic N., Lambrecht N., Respondek M., Peskar B.M., Selective cyclooxygenase-2 inhibitors and their influence on the protective effect of a mild irritant in the rat stomach, Br. J. Pharmacol. 123 (1998) 927–935. [14] Hawkey C.J., Kemp R.T., Walt R.P., Bhaskar N.K., Davies J., Filipowicz B., Evidence that adaptive cytoprotection in rats is not mediated by prostaglandins, Gastroenterology 94 (1988) 948–954. [15] Iseki S., Immunocytochemical localization of cyclooxygenase-1 and cyclooxygenase in the rat stomach, Histochem. J. 27 (1995) 323–328. [16] Kargman S., Charifson S., Cartwiright M., Frank J., Riendean D., Mancini J., Evans J., O’Neill G., Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey and human gastrointestinal tract, Gastroenterology 111 (1996) 445–454. [17] Kennedy B.P., Chan C.C., Culp S.A., Cromlish W.A., Cloning and expression of rat prostaglandin endoperoxide synthase (cyclooxygenase)-2 cDNA, Biochem. Biophys. Res. Commun. 197 (1993) 494–500. [18] Kishimoto Y., Wada K., Nakamoto K., Ashida K., Kamisaki Y., Kawasaki H., Itoh T., Quantitative analysis of cyclooxygenase-2 gene expression on acute gastric injury induced by ischemia-reperfusion in rats, Life Sci. 60 (1997) 127–132. [19] Konturek S.J., Brzozowski T., Piastucki I., Radecki T., Dembinski A., Dembinska-Kiec A., Role of locally generated prostaglandins in adaptive gastric cytoprotection, Dig. Dis. Sci. 27 (1982) 967–971. [20] Konturek P.C., Brzozowski T., Konturek S.J., Dembinski A., Role of epidermal growth factor, prostaglandin and sulfhydryls in stress-induced gastric lesions, Gastroenterology 99 (1990) 1607–1615. [21] Konturek S.J., Brzozowski T., Majka J., Dembinski A., Slomiany A., Slomiany B.L., Transforming growth factor and epidermal growth factor in protection and healing of gastric mucosal injury, Scand. J. Gastroenterol. 27 (1992) 649–655. [22] Konturek S.J., Brzozowski T., Stachura J., Majka J., Role of neutrophils and mucosal blood flow in gastric adaptation to aspirin, Eur. J. Pharmacol. 253 (1994) 107–114. [23] Masferrer I.L., Zweifel B.S., Maming P.T., Hauser S.D., Leahy K.M., Smith W.G., Isakson P.C., Siebert K., Selective inhibition of inducible cyclooxygenase-2 in vivo is antiinflammatory and ulcerogenic, Proc. Natl. Acad. Sci. USA 91 (1994) 3228–3232.
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[24] Masferrer J.L., Isakson P.C., Seibert K., Cyclooxygenase-2 inhibitors, Gastroenterol. Clin. N. Am. 25 (1996) 363–372. [25] Mizuno H., Sakamoto Ch., Matsuda K., Wada K., Kchida T., Noguchi H., Akamatsu T., Kasuga M., Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice, Gastroenterology 12 (1997) 387–397. [26] Nakano O., Sakamoto C., Matsudo K., Konda Y., Matozaki T., Nishisaki H., Wada K., Suzuki T., Uchida T., Nagao M., Induction of cyclooxygenase protein and stimulation of prostaglandin E2 in cultured guinea pig gastric mucous cells, Dig. Dis. Sci. 40 (1995) 1679–1686. [27] Nanji A.A., Miao L., Thomas P., Rahemtullo A., Khwaja S., Zhao S., Peters D., Tahan S.R., Dannenberg A.J., Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat, Gastroenterology 112 (1997) 943–951. [28] Nudel N., Zakut R., Shani M., Neuman S., Levy S., Vaffe D., The nucleotide sequence of the rat cytoplasmic beta-actin gene, Nucleic Acids Res. 11 (1983) 1759–1771. [29] Rainsford K.D., Mechanism of gastrointestinal damage by NSAIDs, Agents Actions 44 (Suppl.) (1993) 59–64. [30] Robert A., Cytoprotection by prostaglandins, Gastroenterology 77 (1979) 761–767. [31] Robert A., Nezamis J.E., Lancaster C., Davis J.P., Field S.O., Hanchar A.J., Mild irritants prevent gastric necrosis through adaptive cytoprotection mediated by prostaglandins, Am. J. Physiol. 245 (1983) 113–121. [32] Svanes K., Gislason H., Guttu K., Herfjord J.K., Ferang J., Gronbech J.E., Role of blood flow in adaptative protection of the cat gastric mucosa, Gastroenterology 100 (1991) 1249–1258. [33] Tarnawski A., Wahlstrom K., Gergely H., Sarfeh I.J., Expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) in normal gastric mucosa. Effect of antacid (hydrotalcit) treatment, Gastroenterology 110 (1996) A275. [34] Vane J.R., Botting R.M., New insights into the mode of action of anti-inflammatory drugs, Inflammation 44 (1995) 1–10. [35] Wallace J.L., Increased resistance of the rat gastric mucosa to hemorrhagic damage after exposure to an irritant. Role of the mucoid cap and prostaglandin synthesis, Gastroenterology 94 (1988) 22–32. [36] Xie W., Chipman J.G., Robertson D.L., Erikson D.L., Simmons D.L., Expression of a mitogen responsive gene encoding prostaglandin synthase is regulated by mRNA splicing, Proc. Natl. Acad. Sci. USA 88 (1991) 2692–2696. [37] Xie W., Robertson D.L., Simmons D.L., Mitogen-inducible prostaglandin G/H synthase: A new target for nonsteroidal antiinflammatory drugs, Drug Dev. Res. 25 (1992) 249–265. [38] Yoshimura H., Kan N., Ogawa N., Preventive and curative effects of prostaglandins on stress ulcer in rats. Application of endoscopic observation, Dig. Dis. Sci. 34 (1989) 436–444.
Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress Tomasz Brzozowskia, Peter Ch. Konturekb, Stanislaw J. Kontureka*, Danuta Drozdowicza, Robert Pajdoa, Michat Pawlika, Iwona Brzozowskaa, Eckhart G. Hahnb a
Department of Physiology, Jagiellonian University School of Medicine, Cracow, Poland b Department of Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany
Abstract — Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg⋅kg–1 i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg⋅kg–1 i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg⋅kg–1 i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg⋅kg–1 i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5–30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg⋅kg–1 i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS, whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 µg⋅kg–1 i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors. © 2000 Elsevier Science Ltd. Published by Éditions scientifiques et médicales Elsevier SAS adaptive cytoprotection / stress damage / mild stress / cyclooxygenase-1 / cyclooxygenase-2 / prostaglandin E2 / gastric blood flow / NS-398 / meloxicam
1. Introduction Endogenous prostaglandins (PG) play a crucial role in the maintenance of gastric mucosal integrity and contribute to gastroprotection and short or long-term adaptation of gastric mucosa to the damaging action of topical irritants [2, 19, 22, 30, 31]. Short-term adaptation known as adaptative cytoprotection was originally introduced by Robert et al. [30, 31] to describe the protective action of certain mild irritants such as 20% ethanol, 5% NaCl and 5 mM taurocholate against the damage induced by these agents applied intragastrically in larger concentrations. This action of mild irritants has been attributed to the protective action of endogenous PG [19, 31] but recently the importance of other protective mediators such as nitric oxide (NO) or sensory nerves were implicated in this phenomenon [7, 14, 32]. * Corresponding author
It is generally accepted that at least two isoforms of the principle enzyme responsible for PG synthesis cyclooxygenase (COX) exist; constitutive COX-1 that provides PG involved in physiological reactions and inducible COX-2 that can be induced by various cytokines, growth factors and endotoxins [6, 9, 36]. PG derived from COX-1 were shown to be important in the maintenance of mucosal integrity, whereas products of the COX-2 have been implicated in inflammatory reactions [6, 9]. Exogenous PG were shown to protect gastric mucosa against stress lesions [38] but it remains unknown which COX is involved in adaptive cytoprotection induced by mild stress against severe stress. Mild stress was shown to prevent gastric lesions induced by severe stress and this effect was attributed to the activity of the adrenomedullary system [12] but the possibility that PG derived from COX-2 are involved in this adaptation has not been examined. In this study, selective COX-1 and COX-2 inhibitors
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were used to determine the contribution of the products of these enzymes in gastric blood flow (GBF) and mucosal protective action of mild stress against gastric lesions induced by stronger stress. In addition, we attempted to assess the expression of COX-1 and COX-2 by RT-PCR in intact gastric mucosa and that exposed to severe stress with or without pretreatment with mild stress. 2. Material and Methods 2.1. Production of stress lesions Stress lesions were provoked by placing animals into individual Bollman cages to produce restrain and immersing them into a water bath for 3.5 h at 23 °C to the level of the xyphoid as described in details in our previous studies [1, 20]. Mild stress was also induced by water immersion and restraint but for a short time starting from 5 min up to 2 h before the subsequent exposure to a stronger stress lasting 3.5 h in order to examine which period of stress would give optimal protective effect against severe WRS. Several groups of rats were exposed to mild WRS at 23 °C followed 1 h later by 3.5 h of WRS with or without the pretreatment with: 1) vehicle (saline); 2) N-[2(cyclohexyloxy)-4-nitrofenyl]-methanesulphonamide (NS-398) (10 mg⋅kg–1 i.g.), a highly selective COX-2 inhibitor [11]; 3) resveratrol (10 mg⋅kg–1 i.g.), a selective COX-1 inhibitor; 4) meloxicam (2 mg⋅kg–1 i.g.), an agent that was shown to inhibit preferentially COX-2 over COX-1 [8, 16, 34]; and 5) indomethacin (5 mg⋅kg–1 i.p.), a non-selective inhibitor of COX. At the dose used in the present study, indomethacin has been shown previously to inhibit gastric PGE2 synthetic capacity by ∼ 90% without causing mucosal damage [21]. The dose of NS-398 was selected on the basis of previous studies showing that it blocks completely PGE2 production in the exudate of air-pouch inflammation but does not inhibit gastric PGE2 content [23]. None of the other COX inhibitors used in this study by themselves produced gastric lesions at the dose tested. Several groups of rats consisting of 6–8 animals received the following treatments: 1) vehicle followed 30 min later by exposure to standard 3.5 h of WRS; 2) mild WRS (5 min–2 h) followed 1 h later by standard WRS; 3) indomethacin (5 mg⋅kg–1 i.p.) followed 90 min later by 3.5 h WRS; 4) NS-398 (10 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 5) resveratrol (5 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 6) meloxicam (2 mg⋅kg–1 i.g.) followed 90 min later by 3.5 h WRS; 7) indomethacin (5 mg⋅kg–1 i.p.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS; 8) NS-398 (10 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and then followed 60 min
later by 3.5 h WRS; 9) resveratrol (5 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS; and finally 10) meloxicam (2 mg⋅kg–1 i.g.) followed 30 min later by mild WRS and 60 min later by 3.5 h WRS. NS-398 was dissolved in absolute ethanol (5 mg⋅mL–1) and was further diluted in saline containing 0.2% methylocelulose and administered in a total volume of 1 mL. Control rats received the corresponding vehicle. In another group of animals treated with COX-1 and COX-2 inhibitors before the combination of mild WRS plus severe WRS, the effect of prostaglandin replacement therapy with 16,16 dimethyl PGE2 (Upjohn, Kalamazoo, MI, USA) was determined. For this purpose, a synthetic PGE2 analogue was administered at a dose of 1 µg⋅kg–1 i.p. 15 min before treatment with each COX-1 or COX-2 inhibitor followed 30 min later by mild WRS for standard 15 min and then challenged 60 min later with 3.5 h WRS. 2.2. Determination of gastric blood flow (GBF) and mucosal generation of PGE2 To evaluate the influence of mild WRS against more severe stress without or with the pretreatment with COX-1 and COX-2 inhibitors, the rats were lightly anaesthetized with ether and the abdomen was opened by a midline incision. The stomach was exposed to assess the GBF using H2-gas clearance technique as described previously [3]. Briefly, the double electrodes of a electrolytic regional blood flowmeter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the mucosa. One of these electrodes was used for the local generation of gaseous H2 and another for the measurement of tissue H2. With this method, the H2 generated locally is carried out by the blood stream, while the polarographic current detector reads out decreasing tissue H2. The clearance curve of tissue H2 was used to calculate an absolute flow rate (mL⋅min–1⋅100 g–1) in the oxyntic gland area as described previously [2, 3]. The measurements were made in three areas of the gastric oxyntic mucosa and the mean values of these measurements were calculated and expressed as percent changes from those recorded in intact animals. For the PGE2 measurement, the biopsy samples of the oxyntic mucosa were taken (about 100 mg) immediately after the animals had been killed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [21]. Briefly, the mucosal sample was placed in preweighed Eppendorf vials and 1 mL of Tris buffer (50 mM, pH 9.6) was added to each vial. The samples were finely minced (about 15 s) with scissors, then washed and centrifuged for 10 s, the pellet being resuspended again in 1 mL Tris. Then each sample was incubated
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on a Vortex mixer (Unipan, Warsaw, Poland) for 1 min and centrifuged for 15 s. The pellet was weighed, and the supernatant was transferred to a second Eppendorf vial containing indomethacin (10 mM) and kept at –20 °C until RIA. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The capability of the mucosa to generate PGE2 was expressed in nanograms per gram of wet tissue weight. 2.3. Expression of COX-1 and COX-2 mRNA transcripts in the gastric mucosa determined by RT-PCR COX-1 and COX-2 mRNA were determined by RT-PCR in the gastric mucosa of intact rats or those exposed to severe WRS with or without the pretreatment with mild WRS applied during a standard period of 15 min. Samples of the gastric oxyntic mucosa (about 500 mg) were scraped off on ice using a glass slide and then immediately snapped frozen in liquid nitrogen, and stored at –80 °C. Total RNA was isolated from the gastric oxyntic mucosa according to Chomczynski and Sacchi [4] using a rapid guanidinum isothiocyanate/phenol chloroform single step extraction kit from Stratagene (Stratagene GmbH, Heidelberg, Germany). Following precipitation, the RNA was resuspended in RNase-free TE buffer and the concentration was estimated by absorbance at 260 nm wavelength. Samples were frozen at –80 °C until analysis. First strand cDNA was synthesized from total cellular RNA (5 µg) using 200 U Strata Script TM reverse transcriptase and oligo (dT) primers (Stratagene GmbH, Heidelberg, Germany). After the reverse transcription, the transcriptase activity was destroyed by heating, and the cDNA were stored at –20 °C until PCR. The primers for COX-1 and COX-2 were synthesized by Biometra (Göttingen, Germany). The primer sequences were designed according to the published cDNA sequence for the rat cyclooxygenases [9, 17, 28]. The COX-1 primer sequences were as follows: upstream, 5’-AGC CCC TCA TTC ACC CAT CAT TT; downstream, 5’-CAG GGA CGC CTG TTC TAC GG. The expected length of this PCR product was 561 bp. The COX-2 primer sequences were as follows: upstream, 5’-ACA ACA TTC CTT CCT TC; downstream, 5’-CCT TAT TTC CTT TCA CAC C. The expected length of this PCR product was 201 bp. Concomitantly, amplification of control rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on the same samples to verify RNA integrity. The primer sequences for GAPDH were purchased from Clontech (Heidelberg, Germany). Reaction mixtures for PCR contained cDNA templates, 50 pmol of each primer, and 2.5 U of Taq DNA
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polymerase (Serva GmbH, Heidelberg) in 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTPs in a volume of 50 µL. RT blanks (no RNA included) and PCR blanks (no cDNA products included) were included in each analysis. DNA amplification was carried out under the following conditions; denaturation at 94 °C for 1 min, annealing at 60 °C for 45 s and extension at 72 °C for 45 s. To maximize amplification specificity, Taq DNA polymerase was added to the PCR mixture during the hot start of cycle 1. The COX-1 and COX-2 genes were amplified for 30 cycles. Each PCR-product (8 µL) was electrophoresed on a 1.5% agarose gel stained with ethidium bromide, and then visualized under UV light. Location of predicted PCR product was confirmed by using a 100-base pair ladder (Gibco BRL/Life Technologies, Eggenstein, Germany) as standard marker. To avoid PCR contamination, PCR reactions were prepared in a dedicated area used only for PCR and the PCR product were opened in a laminar flow hood separated from the PCR preparation area.
3. Results 3.1. Stress-induced gastric lesions and demonstration of adaptive cytoprotection by mild stress As shown in figure 1, exposure of rats to a standard 3.5 h WRS produced numerous gastric lesions (approximately 18–22 per stomach) that were identified predominantly in the oxyntic mucosa as bleeding erosions. In contrast, mild WRS lasting from 5 to 30 min by itself failed to produce gross gastric lesions (data not shown) but significantly reduced the lesions provoked by more severe standard stress, the optimal protective effect being obtained with 15 min mild WRS (figure 1). In further studies, mild WRS lasting for 15 min was used and it caused about 80% reduction in lesions induced by standard severe stress. The gross appearance of such stomach with the lesions induced by the exposure to 3.5 h WRS with or without pretreatment with 15 min mild WRS is presented in figure 2 (lower), BAs shown in figure 2 (upper), the pretreatment with mild WRS for 15 min significantly attenuated gross mucosal lesions induced by more severe stress. Extension of the duration of mild WRS to 30 min was not more effective in reduction of lesions induced by standard stress when compared to that recorded in animals subjected to 15 min mild WRS (figure 1). Further extension of the duration of WRS up to 1 h did not result in any reduction in the number of gastric lesions caused by standard severe stress, while the application of 2 h of WRS aggravated lesions induced by the subsequent exposure to this standard severe stress (figure 1).
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Figure 1. Mean number of gastric lesions and gastric mucosal blood flow (GBF) in rats exposed to 3.5 h water immersion and restraint stress (WRS) without or with pretreatment with mild WRS ranging from 5 to 120 min. Results are mean ± SEM of 8 rats. Asterisk indicates a significant change (P < 0.05) as compared to the value obtained in rats without pretreatment with mild WRS. Cross indicates a significant change as compared to animals pretreated with mild WRS for 15 min followed by exposure to 3.5 h of more severe stress. Double cross indicates a significant change as compared to the value recorded in gastric mucosa exposed to severe WRS.
GBF in intact gastric mucosa averaged 46 ± 6 mL⋅min–1⋅100 g–1 tissue (taken as 100%) and this was reduced by about 49% in rats subjected to 3.5 h of severe stress (figure 1). Application of mild WRS before a standard 3.5 h severe stress attenuated the fall of GBF caused by this stress and the maximal increase of GBF was reached in animals pretreated with 15 min of mild WRS (figure 1). With the prolongation of mild WRS to 1 h before more severe stress, the fall in GBF was not significantly different from that recorded with standard WRS but further extension of the mild WRS up to 2 h caused a fall in GBF greater than that observed with severe stress alone (figure 1). 3.2. Effect of suppression of PGE2 generation on gastroprotection induced by mild WRS The generation of PGE2 in the intact gastric mucosa averaged 148 ± 12 ng⋅g–1 of wet tissue weight but when the mild WRS was applied alone (for 15 min), a significant increase in PGE2 generation was recorded, which reached the value of 193 ± 8 ng⋅g–1 of wet tissue weight. Such standard mild WRS, which by itself failed to produce gastric lesions, resulted in a significant increase in the GBF by about 23% as compared to that in intact gastric mucosa (data not
Figure 2. Gross appearance of the rat gastric mucosa after the exposure to 3.5 h of stress without or with pretreatment with mild WRS. In rat exposed to 3.5 h of severe stress, numerous gastric lesions are evident (lower panel), whereas in rat pretreated with standard 15 min of mild WRS before the exposure to severe stress, the number of gastric lesions is markedly attenuated (upper panel).
shown). In contrast, the exposure to 3.5 h WRS alone which caused gastric lesions, attenuated significantly the GBF and PGE2 generation as compared to those in the intact mucosa. Pretreatment with mild WRS significantly reduced gastric lesions induced by more severe stress and attenuated the decrease in GBF and PGE2 generation induced by this severe stress (figure 3). The administration of indomethacin (5 mg⋅kg–1 i.p.) 90 min before the application of 3.5 h of stress, suppressed mucosal generation of PGE2 by about 90% and increased significantly the mean number of gastric lesions, the effect being accompanied by a significant fall in the GBF (figure 3). When indomethacin was applied before the combination of mild WRS plus severe WRS, it abolished completely the reduction in gastric lesions and accompanying increase in the GBF induced by the pretreatment with mild WRS (figure 3).
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Figure 3. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with indomethacin (5 mg·kg–1 i.p.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa pretreated with mild WRS before exposure to 3.5 h severe stress.
Figure 4. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with resveratrol (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals pretreated with standard mild WRS before exposure to 3.5 h severe stress.
3.3. Effect of specific and non-specific COX-1 and COX-2 inhibitors on adaptive cytoprotection induced by mild WRS against severe stress
meloxicam, applied i.p. in a dose of 2 mg⋅kg–1, aggravated significantly gastric lesions induced by 3.5 h severe stress and decreased significantly the GBF recorded in animals exposed to this severe stress (figure 5). Meloxicam, which suppressed the PGE2 generation, abolished completely the protective and hyperemic effects of mild 15 min WRS on subsequent more severe stress (figure 5).
Figure 4 shows the effects of pretreatment with resveratrol (10 mg⋅kg–1 i.g.), a specific inhibitor of COX-1, on the gastric lesions induced by severe stress with or without pretreatment with mild WRS. Pretreatment with resveratrol, which produced small and not significant decrease in the PGE2 generation, failed to influence significantly the number of lesions caused by exposure to 3.5 h severe stress and accompanying fall in the GBF. Such pretreatment with resveratrol reversed, in part, the mild WRS-induced gastric protection and accompanying increase in the GBF and attenuated significantly the rise in the PGE2 caused by the mild WRS (figure 4). Pretreatment with NS-398, a specific inhibitor of COX-2 given in a dose of 10 mg⋅kg–1 (i.g.), failed to affect significantly the number of gastric lesions induced by severe stress (3.5 h) and accompanying fall in GBF but significantly attenuated the protection against this stress induced by 15 min mild WRS and completely reversed the rise in GBF evoked by mild WRS (figure 5). Such pretreatment with NS-398 failed to influence significantly the PGE2 generation in rats exposed to severe stress with or without the pretreatment with mild WRS (figure 5). Pretreatment with
3.4. Effect of PG replacement therapy with exogenous PGE2 on adaptive cytoprotection induced by mild WRS As shown in figure 6, the pretreatment with COX-1 and COX-2 inhibitors before the combination of mild WRS plus severe stress produced similar alterations in the number of gastric lesions and GBF as presented in figures 3, 4. Administration of PGE2 (1 µg⋅kg–1 i.p.) counteracted, in part, the inhibitory effect of nonselective COX inhibitors on the mild WRS-induced protection against severe stress and accompanying alterations in GBF (figure 6). Such pretreatment with PGE2 completely abolished the increase in the number of lesions and the accompanying fall in GBF induced by administration of the selective COX-2-inhibitor, NS-398, or selective COX-1-inhibitor, resveratrol (figure 6).
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Figure 5. Mean lesion number, GBF and mucosal generation of PGE2 in the gastric mucosa of rats exposed to the combination of mild WRS (for 15 min) plus 3.5 h of severe stress without or with the pretreatment with meloxicam (2 mg·kg–1 i.p.) or NS-398 (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa of rats pretreated with mild WRS before exposure to 3.5 h severe stress.
3.5. Expression of COX-1 and COX-2 mRNA by RTPCR in gastric mucosa of rats exposed to vehicle or severe stress with or without the pretreatment with mild WRS Figure 7A–C shows the mRNA expression of β-actin, COX-1 and COX-2 in the gastric mucosa of control rats treated with vehicle or those exposed to 3.5 h stress alone, mild WRS alone or their combination. The expression of mRNA for β-actin was wellpreserved in the mucosal samples taken from rats treated with vehicle (control) or those exposed to severe stress with or without the pretreatment with mild WRS (figure 7A). The COX-1 mRNA was detectable in the vehicle-treated gastric mucosa as well as in the mucosa exposed to severe stress with or without pretreatment with mild WRS (figure 7B). In contrast, COX-2 mRNA was undetectable in vehicle control animals but has been traced in rats exposed to mild WRS alone or severe stress with or without the combination with mild WRS (figure 7C). 4. Discussion In the present study, we confirmed that the exposure of rats to mild WRS protects the stomach against
Figure 6. Mean lesion number and the GBF in the gastric mucosa of rats exposed to standard 15 min mild WRS followed by exposure to 3.5 h of severe stress without or with the pretreatment with 16,16 dimethyl PGE2 (1 µg·kg–1 i.p.) added to indomethacin (5 mg·kg–1 i.p.), meloxicam (2 mg·kg–1 i.p.), resveratrol (10 mg·kg–1 i.g.) or NS 398 (10 mg·kg–1 i.g.). Results are mean ± SEM of 8 rats. Asterisk indicates a significant change as compared to the value obtained in vehicle-control rats exposed to 3.5 h of severe stress. Cross indicates a significant change as compared to the value obtained in animals exposed to severe stress without pretreatment with mild WRS. Double cross indicates a significant change as compared to the value recorded in gastric mucosa of rats treated with mild WRS plus 3.5 h of severe stress without pretreatment with a PGE2 analogue.
gastric mucosal lesions provoked by severe stress probably via the phenomenon of adaptive cytoprotection similar to that afforded by mild irritants [19, 30, 31]. However, this protective effect was observed only when shorter periods of mild WRS lasting from 5 to 30 min was employed before the severe stress. An extension of mild WRS up to 2 h not only failed to protect the gastric mucosa but even augmented the stress-induced gastric lesions. Furthermore, such pretreatment with 15 min of mild WRS (which attenuated lesions caused by severe stress) produced significant rise in PGE2 generation and GBF in the gastric mucosa suggesting that this enhancement in the gastric microcirculation was probably due to increased generation of mucosal PG. This is supported by the fact that pretreatment with indomethacin, a non-selective inhibitor of COX, which strongly suppressed PGE2 generation, completely abolished the protective and hyperemic effects of mild WRS against lesions provoked by severe stress. In contrast, the administration of NS-398, a specific COX-2 inhibitor, or resveratrol, a specific COX-1 inhibitor, reversed only in part the gastroprotection and accompanying rise in GBF induced by mild WRS and these effects were fully restored by the addition of exogenous PGE2 to this
COX-1 and -2 in adaptive cytoprotection
Figure 7. Analysis of reverse transcriptase polymerase chain reaction (RT-PCR)-amplified β-actin, COX-1 and COX-2 products using specific primers. M = PCR size marker (PhiX 174 RF DNA Hae III Digest, New England Biolabs, USA); lane 1, intact gastric mucosa; lane 2, 3.5 h stress alone; lane 3, standard mild WRS alone; lane 4, standard mild WRS plus 3.5 h severe stress.
COX-2 or COX-1 inhibitor. Moreover, the strong signals for COX-1 mRNA was detected in the vehicle treated control gastric mucosa as well as in the mucosa of rats exposed to mild WRS, severe stress or their combination. In contrast, COX-2 mRNA was undetectable in the intact vehicle-treated gastric mucosa but was well-defined in the mucosa of animals exposed to mild WRS or severe stress with or without pretreatment with mild WRS suggesting that the upregulation of COX-2 mRNA with excessive production of protective PG could contribute to adaptive cytoprotection induced by mild WRS. Previous studies have established that PG synthesis depends upon the activity of cyclooxygenase (COX), a rate limiting enzyme in the synthesis of eicosanoids [6, 34]. Two isoforms of COX were identified in many cells; a constitutive enzyme designated as COX-1 and inducible isoform known as COX-2 [17, 24]. Under
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physiological conditions, prostanoid synthesis depends upon the availability of arachidonic acid and the enzymatic activity of COX-1, which is a major target for NSAIDs resulting in mucosal damage in the stomach [29]. In contrast, COX-2 is not constitutively expressed in most of the tissues but could be induced by a variety of stimuli including interleukin-1, tumour necrosis factor (TNF)-α, growth factors, lipopolisacharide and oxidative stress [24, 26, 37]. Downregulation of COX-1 associated with a decrease of cytoprotective prostanoids was recently implicated in the exacerbation of liver injury by ethanol [27]. On the other hand, the upregulation of COX-2 due to ethanol treatment in that study [27] resulted in increased synthesis of inflammatory and vasoactive eicosanoids. We confirmed our previous observations [1, 20] that exposure of the rats to 3.5 h stress induced by cold and restraint produces gastric lesions and that this effect is accompanied by a decrease in PGE2 generation and a marked fall in GBF. Non-topical irritant such as mild WRS, which by itself failed to produce gastric lesions, or alter increased PGE2 generation, prevented the formation of lesions induced by severe stress, supporting previous findings with mild irritants [35] that endogenous PG are essential for the development of adaptive cytoprotection. We attempted in this study to determine the relative contribution of COX-1 and COX-2 to stress ulcerogenesis and adaptive cytoprotection induced by mild WRS. In agreement with previous studies [6, 36, 37], we found that in the normal stomach COX-1 is predominantly expressed, whereas COX-2 mRNA is not. Strong signals for COX-1 mRNA were also detected in gastric mucosa of rats exposed to mild or severe WRS and in those pretreated with mild WRS before exposure to severe stress. It is of interest that an overexpression of COX-2 mRNA was observed in the mucosa exposed to mild or severe stress applied alone or in combination, suggesting that the upregulation of the gene for COX-2 may contribute to the protective action of mild WRS. The overexpression of COX-2 mRNA during severe stress might be due to the deficit of PGE2 generation in the gastric mucosa as demonstrated in the present study. This notion is in keeping with recent finding by Davies et al. [5], who found an upregulation of COX-2 mRNA in a stomach acutely treated with aspirin applied at a dose that induced gastric damage and almost complete suppression of mucosal generation of PGE2. It was concluded [5] that this elevation in COX-2 expression was not a specific response to injury because topically applied irritants such as ethanol, indomethacin or salicylate failed to alter significantly both, COX-2 mRNA and COX-2 protein expression. In contrast to this observation, Mizuno et al. [25] showed that COX-2 mRNA and COX-2 protein levels in mice stomach were markedly
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increased after ethanol-induced acute mucosal injury and during the healing of chronic gastric ulcers. As mentioned before, the upregulation of COX-2 mRNA observed in our present study could be triggered by the depletion of endogenous PG caused by severe stress. This is supported by the observation that replacement therapy with exogenous PGE2 added to specific COX-2 inhibitor to cover the deficiency of PG, attenuated significantly the deleterious effect of this inhibitor. In another study, the increased expression of COX-2 mRNA induced by aspirin at a dose that probably suppressed mucosal generation of PGE2, was significantly diminished by the administration of exogenous PGE2 [5]. Thus, our study demonstrates that PG derived from both COX-1 and COX-2 enzymes seem to mediate the protective response to mild stressor. This is supported by the fact that non-specific inhibitor of COX, indomethacin at the dose that suppressed PGE2 generation at about 90%, completely abolished the protective and hyperemic response to mild WRS. It is of interest, that meloxicam, shown to inhibit preferentially COX-2 but also inhibiting COX1 [8], antagonized almost completely these responses in the manner similar to that observed with indomethacin. In contrast, NS-398 which failed to alter PGE2 generation in gastric mucosa significantly attenuated the protection and accompanying hyperemia induced by mild WRS, and a similar degree of reversal of the protective effect of mild WRS was obtained in animals pretreated with specific COX-1 inhibitor, resveratrol. Thus, it is reasonable to assume that this adaptive cytoprotection could be completely antagonized by agents that suppress both COX-1 and COX-2 pathways or impaired by inhibition of either, COX-1 or COX-2 pathway. This notion is in keeping with recent observations by Gretzer et al. [13] that PG generated by COX-2 are involved in adaptive cytoprotection induced by topically applied mild irritant. These authors [33] concluded that COX-2 is constitutively expressed and may play a significant role in adaptive cytoprotection induced by topically acting mild irritant. In agreement with this notion, the COX-2 mRNA and COX-2 protein were recently identified in endothelia of gastric mucosal microvessels [33]. COX-1 is generally regarded to be a constitutively expressed enzyme, whereas COX-2 is usually referred to as the inducible enzyme isoform [16]. However, COX-2 was shown to be constitutively expressed in some tissues and there is evidence of the inducibility of COX-1 in gastric mucosa of rats treated chronically with endotoxin [10]. It is of interest that immunoreactivity of COX-2 was detected in the prepyloric and fundic regions of the rat gastric mucosa suggesting that COX-2 could be expressed in unstimulated stomach [15]. We were not able to show expression of COX-2 mRNA in intact animals similarly to a recent
report [10] but an overexpression of COX-2 mRNA was observed in rats exposed to mild and severe stress or after their combination suggesting that in addition to COX-1, COX-2-derived prostaglandins play an important role in adaptive cytoprotection induced by mild WRS. This is in keeping with the finding that the induction of COX-2 was triggered by ischemiareperfusion and accompany the healing of chronic gastric ulcerations thus indicating that COX-2-derived PG are essential to ensure ulcer healing [18, 25]. On the other hand, that COX-1 was the primary source of prostaglandins responsible for this mild stress is supported by our finding that indomethacin, which is more selective for COX-1 than COX-2 [16, 34], reversed completely the protection induced by mild WRS against severe stress, whereas NS-398, a highly selective COX-2 inhibitor in the same experimental conditions, had only partial inhibitory effect on the stress lesions and failed to influence accompanying enhancement in PGE2 generation induced by mild WRS. References [1]
Brzozowski T., Konturek S.J., Majka J., Dembinski A., Drozdowicz D., Epidermal growth factor, polyamines and prostaglandins in healing of stress induced gastric lesions in rats, Dig. Dis. Sci. 38 (1993) 276–283. [2] Brzozowski T., Konturek P.Ch., Konturek S.J., Stachura J., Gastric adaptation to aspirin and stress enhances gastric mucosal resistance against the damage by strong irritants, Scand. J. Gastroenterol. 31 (1996) 118–125. [3] Brzozowski T., Konturek S.J., Sliwowski Z., Pajdo R., Drozdowicz D., Stachura J., Role of β-adrenoceptors in gastric mucosal integrity and gastroprotection induced by epidermal growth factor, Digestion 58 (1997) 319–331. [4] Chomczynski P., Sacchi N., Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. [5] Davies N.M., Sharkley K.A., Asfaha S., MacNaughton W.K., Wallace J.L., Aspirin causes rapid up-regulation of cyclooxygenase-2 expression in the stomach of rats, Alim. Pharmacol. Ther. 11 (1997) 1101–1108. [6] Eberhart C.E., Dubois R.N., Eicosanoids and the gastrointestinal tract, Gastroenterology 109 (1995) 285–301. [7] Endo K., Kao J., Bomek M.J., Leung F.W., Mechanism of gastric hyperemia induced by intragastric hypertonic saline in rats, Gastroenterology 104 (1993) 114–121. [8] Engelhardt G., Bogel R., Schmitzer Ch., Utzman R., Meloxicam; Influence on arachidonate metabolism. Part 1 in vitro findings, Biochem. Pharmacol. 51 (1996) 21–28. [9] Feng L., Xia Y., Garcia G.E., Hwang D., Wilson C.B., Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha and lipopolysaccharide, J. Clin. Invest. 95 (1995) 1669–1675. [10] Ferraz J.G., Sharkey K.A., Reuter B.K., Asfaha S., Tigley A.W., Brown M.L., McKnight W., Wallace J.L., Induction of cyclooxygenase-1 and -2 in the rat stomach during endotoxemia: role in resistance to damage, Gastroenterology 3 (1997) 195–204.
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[11] Futaki N., Yoshikawa K., Hamasaka Y., Arai I., Higuchi S., Izuka H., Otomo S., NS-398 a novel non-steroidal antiinflammatory drug with potent analgesic and antipyretic effects which causes minimal stomach lesions, Gen. Pharmacol. 24 (1993) 105–110. [12] Glavin G.B., Lockhart L.K., Rockman G.E., Hell A.M., Kierhan K.M., Evidence of cross-stressor induced adaptive gastric cytoprotection, Life Sci. 41 (1987) 2223–2227. [13] Gretzer B., Ehrlich K., Maricic N., Lambrecht N., Respondek M., Peskar B.M., Selective cyclooxygenase-2 inhibitors and their influence on the protective effect of a mild irritant in the rat stomach, Br. J. Pharmacol. 123 (1998) 927–935. [14] Hawkey C.J., Kemp R.T., Walt R.P., Bhaskar N.K., Davies J., Filipowicz B., Evidence that adaptive cytoprotection in rats is not mediated by prostaglandins, Gastroenterology 94 (1988) 948–954. [15] Iseki S., Immunocytochemical localization of cyclooxygenase-1 and cyclooxygenase in the rat stomach, Histochem. J. 27 (1995) 323–328. [16] Kargman S., Charifson S., Cartwiright M., Frank J., Riendean D., Mancini J., Evans J., O’Neill G., Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey and human gastrointestinal tract, Gastroenterology 111 (1996) 445–454. [17] Kennedy B.P., Chan C.C., Culp S.A., Cromlish W.A., Cloning and expression of rat prostaglandin endoperoxide synthase (cyclooxygenase)-2 cDNA, Biochem. Biophys. Res. Commun. 197 (1993) 494–500. [18] Kishimoto Y., Wada K., Nakamoto K., Ashida K., Kamisaki Y., Kawasaki H., Itoh T., Quantitative analysis of cyclooxygenase-2 gene expression on acute gastric injury induced by ischemia-reperfusion in rats, Life Sci. 60 (1997) 127–132. [19] Konturek S.J., Brzozowski T., Piastucki I., Radecki T., Dembinski A., Dembinska-Kiec A., Role of locally generated prostaglandins in adaptive gastric cytoprotection, Dig. Dis. Sci. 27 (1982) 967–971. [20] Konturek P.C., Brzozowski T., Konturek S.J., Dembinski A., Role of epidermal growth factor, prostaglandin and sulfhydryls in stress-induced gastric lesions, Gastroenterology 99 (1990) 1607–1615. [21] Konturek S.J., Brzozowski T., Majka J., Dembinski A., Slomiany A., Slomiany B.L., Transforming growth factor and epidermal growth factor in protection and healing of gastric mucosal injury, Scand. J. Gastroenterol. 27 (1992) 649–655. [22] Konturek S.J., Brzozowski T., Stachura J., Majka J., Role of neutrophils and mucosal blood flow in gastric adaptation to aspirin, Eur. J. Pharmacol. 253 (1994) 107–114. [23] Masferrer I.L., Zweifel B.S., Maming P.T., Hauser S.D., Leahy K.M., Smith W.G., Isakson P.C., Siebert K., Selective inhibition of inducible cyclooxygenase-2 in vivo is antiinflammatory and ulcerogenic, Proc. Natl. Acad. Sci. USA 91 (1994) 3228–3232.
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[24] Masferrer J.L., Isakson P.C., Seibert K., Cyclooxygenase-2 inhibitors, Gastroenterol. Clin. N. Am. 25 (1996) 363–372. [25] Mizuno H., Sakamoto Ch., Matsuda K., Wada K., Kchida T., Noguchi H., Akamatsu T., Kasuga M., Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice, Gastroenterology 12 (1997) 387–397. [26] Nakano O., Sakamoto C., Matsudo K., Konda Y., Matozaki T., Nishisaki H., Wada K., Suzuki T., Uchida T., Nagao M., Induction of cyclooxygenase protein and stimulation of prostaglandin E2 in cultured guinea pig gastric mucous cells, Dig. Dis. Sci. 40 (1995) 1679–1686. [27] Nanji A.A., Miao L., Thomas P., Rahemtullo A., Khwaja S., Zhao S., Peters D., Tahan S.R., Dannenberg A.J., Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat, Gastroenterology 112 (1997) 943–951. [28] Nudel N., Zakut R., Shani M., Neuman S., Levy S., Vaffe D., The nucleotide sequence of the rat cytoplasmic beta-actin gene, Nucleic Acids Res. 11 (1983) 1759–1771. [29] Rainsford K.D., Mechanism of gastrointestinal damage by NSAIDs, Agents Actions 44 (Suppl.) (1993) 59–64. [30] Robert A., Cytoprotection by prostaglandins, Gastroenterology 77 (1979) 761–767. [31] Robert A., Nezamis J.E., Lancaster C., Davis J.P., Field S.O., Hanchar A.J., Mild irritants prevent gastric necrosis through adaptive cytoprotection mediated by prostaglandins, Am. J. Physiol. 245 (1983) 113–121. [32] Svanes K., Gislason H., Guttu K., Herfjord J.K., Ferang J., Gronbech J.E., Role of blood flow in adaptative protection of the cat gastric mucosa, Gastroenterology 100 (1991) 1249–1258. [33] Tarnawski A., Wahlstrom K., Gergely H., Sarfeh I.J., Expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) in normal gastric mucosa. Effect of antacid (hydrotalcit) treatment, Gastroenterology 110 (1996) A275. [34] Vane J.R., Botting R.M., New insights into the mode of action of anti-inflammatory drugs, Inflammation 44 (1995) 1–10. [35] Wallace J.L., Increased resistance of the rat gastric mucosa to hemorrhagic damage after exposure to an irritant. Role of the mucoid cap and prostaglandin synthesis, Gastroenterology 94 (1988) 22–32. [36] Xie W., Chipman J.G., Robertson D.L., Erikson D.L., Simmons D.L., Expression of a mitogen responsive gene encoding prostaglandin synthase is regulated by mRNA splicing, Proc. Natl. Acad. Sci. USA 88 (1991) 2692–2696. [37] Xie W., Robertson D.L., Simmons D.L., Mitogen-inducible prostaglandin G/H synthase: A new target for nonsteroidal antiinflammatory drugs, Drug Dev. Res. 25 (1992) 249–265. [38] Yoshimura H., Kan N., Ogawa N., Preventive and curative effects of prostaglandins on stress ulcer in rats. Application of endoscopic observation, Dig. Dis. Sci. 34 (1989) 436–444.