S. KWIECIEÑ, T. BRZOZOWSKI, P.CH. KONTUREK, S.J. KONTUREK

THE ROLE OF REACTIVE OXYGEN SPECIES IN ACTION OF NITRICOXIDE-DONORS ON STRESS-INDUCED GASTRIC MUCOSAL LESIONS

Department of Physiology Jagiellonian University School of Medicine, Cracow, Poland

The experimental model of acute gastritis such as water immersion restraint (WRS)stress-induced gastric injury is useful tool in examination of pathomechanism ofacute gastritis. Nitric oxide (NO) plays an important role in the maintenance ofgastric barrier, however, the interaction between reactive oxygen species (ROS) andNO on gastric mucosal integrity has been little studied. The purpose of our presentstudy was to explain the participation of ROS in healing of WRS-induced gastriclesions accelerated by NO. Experiments were carrying out on 120 male Wistar rats.To assess gastric blood flow (GBF) laser Doppler flowmeter was used and thenumber of gastric lesions was counted in each stomach. The colorimetric assays wereused to determine gastric tissue level of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), the products of lipid peroxidation by ROS, as well assuperoxide dismutase (SOD) activity, the enzyme scavanger of ROS. Wedemonstrated that 3.5 h of WRS resulted in appearance of acute gastric lesionsaccompanied by a significant decrease of GBF. Biological effects of ROS wereestimated by measuring tissue levels of MDA and 4-HNE, as well as the SODactivity. It was demonstrated that 3.5 h of WRS led to significant increase of mucosallevels of MDA and 4-HNE, and it was accompanied by a decrease of SOD activity.Pretreatment with NO-donors (SIN-1, SNAP, nitroglycerin, NO-ASA) resulted inreduction in gastric lesion number, increment of GBF, decrease of MDA and 4-HNEtissue level and increase of SOD activity. Suppression of ROS plays an importantrole in the action of NO-donors on healing of acute gastric lesions induced by 3.5 hof WRS. NO-donors caused an attenuation of lipid peroxidation as documented by adecrease of MDA and 4-HNE levels and enhancement of antioxidative properties asevidenced by an increase of SOD activity.

K e y w o r d s : water immersion restraint stress, MDA, SOD, NO-donors, SIN-1, SNAP,nitroglycerin, NO-ASA

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 4, 761773

www.jpp.krakow.pl

INTRODUCTION

Acute gastric mucosal lesions represent an important clinical problem.Animal models allow to recognize details of pathomechanisms of acute gastricdamage. The model of water immersion and restraint stress (WRS), proposed byTakagi et al. (1), is especially useful in these investigations. This model appearsto be very suitable in testing various factors affecting the formation and healingof gastric mucosal lesions, for example sulphydryls, endogenous prostaglandins(2), growth factors, polyamines (3), etc. The effect of apoptosis on production ofgastric lesions in this model has also been determined (4). Experiments on therole of gastric acid secretion and certain bacterial metabolites, affecting thissecretion such as N-alpha-methylhistamine in acute WRS ulcerogenesis, werecarried out (5).

The gastric barrier protects the mucosa against damage of its deeper structuresby hydrogen ion and other noxious substances originating from the gastric lumen(6). An important role in damage and protection of this barrier is played by gastricmicrocirculation. Disturbances in blood perfusion of gastric mucosa result in theformation of erosions and ulcers. This phenomenon typically occurs inexperimental model of ischemic gastric lesions (7).

The main factors, which regulate gastric blood flow, are prostaglandins,sensory peptides released from endings of afferent nerves and nitric oxide (8, 9).The function of nitric oxide (NO) in the regulation of gastric blood flow (GBF),which participates in maintenance of mucosal integrity, has been a subject ofmany investigations (10-12). These investigations focused on the involvement ofNO in protection of gastric mucosal perfusion as well as on the interaction of NOand prostaglandins on gastric mucosa.

Little information is available regarding the contribution of reactive oxygenspecies (ROS) and NO to the mechanism of gastric mucosal integrity. Previousstudies focused mainly on the participation of ROS in experimental ischemic modelof gastric lesions (13, 14). Results of our previous experiments indicated that 3.5hours of WRS leads to increased oxidative metabolism, comparable with thatobserved in ischemia-reperfusion model of gastric injury. Under these conditions theevaluation of ROS participation in the NO action on gastric mucosal seems to be ofinterest. Two parameters are usually useful for assessment of biological effects ofROS namely the tissue levels of malondialdehyde (MDA) plus 4-hydroxynonenal(4-HNE) and the activity of superoxide dismutase (SOD). Tissue levels of MDA and4-HNE are used as indicators of lipid peroxidation. SOD activity reflects theantioxidative properties of various tissues including gastric mucosa (Fig. 1).

In our experiments acute gastric lesions have been induced by 3.5 hours ofWRS and intragastrical administration of NO-donors was used prior to the WRS.The aim of our present investigations was to elucidate the participation of ROS inhealing of WRS-induced gastric lesions, in tests without and with addition of NO-donors.

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MATERIAL AND METHODS

Experiments were carried out on 120 male Wistar rats, weighing about 200 g and fasted for 24h before all studies. Studies were approved by the Ethic Committee for Animal Research ofJagiellonian University College of Medicine.

Production of gastric lesions

The animals were divided into 6 groups. In first group, rats underwent 3.5 h of WRS intemperature 23°C, using the method originally proposed by Takagi et al. (1). In second group, 3-morpholinosyndnoimine (SIN-1) was administered intragastrically (ig.), using a metal orogastrictube in a dose of 5 mg/kg, given 30 min prior to 3.5 h of WRS. In third group S-nitroso-N-acetyl-DL-penicillamine (SNAP) was applied intragastrically (i.g.) in dose of 5 mg/kg, 30 min prior to 3.5h of WRS. In forth group nitroglycerin (10 mg/kg i.g.) was used 30 min prior to 3.5 h of WRS. Infifth group NO-aspirin (NO-ASA) - 40 mg/kg i.g. - was administered 30 min prior to 3.5 h of WRS.Group sixth of animals served as a control group and did not undergo any procedures (control group).

Determination of gastric blood flow and number of lesions

The gastric lesions were evaluated 3.5 hours after start of WRS. To assess gastric blood flow(GBF) laser Doppler flowmeter (Laserflo, model BPM 403A, Blood Perfusion Monitor,Vasamedics, St. Paul, Minnesota, USA) was used. The animals were anaesthetized with Vetbutal 50mg/kg (Biowet, Pu³awy, Poland), then the abdomen was opened and the stomach was exposed todetermine the GBF. Blood flow was measured on anterior and posterior wall of stomach. The meanvalues of these measurements were calculated and expressed as percent change from value recordedin intact mucosa. To establish the number of gastric lesions computerized planimetry (Morphomat,Carl Zeiss, Berlin, Germany) was used, as described previously (5, 15).

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Fig. 1. Transformation pathways of a superoxide radical anion in the organ tissue. Superoxide ismetabolized via the process of lipid peroxidation or neutralization undergo to form H2O2 and H2O.

Measurement of lipid peroxydation

For determination of lipid peroxydation in tested groups, tissue levels of malondialdehyde(MDA) and 4-hydroxynonenal (4-HNE) were measured and they were used as indicators of lipidperoxidation. The procedure of MDA and 4-HNE determination was as follows: about 600 mg ofgastric mucosa was excised, quickly washed in test tube and 20 ml 0.5 M BHT (butylatedhydroxytoluene) was added in order to prevent sample oxidation. This sample was subsequentlyhomogenized in 20 mM Tris for 15 sec. in pH 7.4. Then homogenate was centrifuged (3000 g at4°C for 10 min) and obtained clear supernatant was stored at -80°C prior to testing.

The colorimetric assay for lipid peroxidation (Bioxytech LPO-586, Oxis, Portland, USA) wasused to determine of MDA and 4-HNE tissue concentration. This assay is based on the reaction ofa chromogenic reagent N-methyl-2-phenylindole with MDA and 4-HNE at 45°C. This reactionyields a stable chromophore with maximal absorbance at 586 nm. This absorbance was measuredby spectrophotometer Marcel s300, Warsaw, Poland. Results were expressed as nanomol per gramof tissue (nmol/g) according to our study published previously (15).

Determination of SOD activity

For the determination of activity of superoxide dismutase (SOD), a sample of gastric mucosa wastaken, as described above. The colorimetric assay for assessment of SOD activity (Bioxytech SOD-525, Oxis, Portland, USA) was used. This method is based on the SOD-mediated increase in the rateof autooxidation of tetrahydrobenzofluorene in aqueous alkaline solution to yield a chromophore

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Fig. 2. Mean number of gastric lesions and gastric blood flow (GBF) after 3.5 h of water immersionrestraint stress (WRS) without or with intragastrical pretreatment (i.g.) with SIN-1 (5 mg/kg i.g.) orSNAP (5 mg/kg i.g.). Results are mean ± SEM of 6-8 rats. Cross (+) indicates significant change,as compared with the value obtained in vehicle control group. Asterisk (*) indicates significantchange, as compared with the values obtained in rats exposed to 3.5 h of WRS alone.

with maximum absorbance at 525 nm. This absorbance was measured by spectrophotometer Marcels300, Warsaw, Poland. Results were expressed as units per gram of tissue (U/g)(15).

Statistical analysis

Results are expressed as means ± SEM. Statistical analysis was done using nonparametricMann-Whitney test. Differences with P

increase of GBF as compared with the 3.5 h of WRS group. After administrationof SIN-1 at a dose of 5 mg/kg (Fig. 2) a decrease of mean lesion number to6.6±1.2 was observed. Administration of nitroglycerin (10 mg/kg i.g.) or NO-ASA (40 mg/kg i.g.) evoked similar effects to those observed with SIN-1, namelya reduction in gastric lesion number to the values of 14.0±1.0, 13.7±1.6,respectively, and a significant rise in the GBF (Fig. 3).

Lipid peroxidation products

Concentration of MDA and 4-HNE in the intact mucosa was very low, near toanalytical limit of detection (5.9±0.05 nmol/g). After 3,5 h of WRS the level ofMDA and 4-HNE increased almost three times, reaching the value of 15.8±1.0nmol/g. Administration of SIN-1 (5 mg/kg i.g.) resulted in a significant decreasein MDA and 4-HNE concentrations as compared to respective values in animalsexposed to the 3.5 h of WRS alone. Application of SNAP (5mg/kg i.g.) producedsignificant decrease of MDA and 4-HNE levels, as compared with those observedin the 3.5 h of WRS group (Fig. 4). Pretreatment with nitroglycerin (10 mg/kgi.g.) or NO-ASA (40 mg/kg i.g.) led to significant fall in lipid peroxide

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Fig. 4. Concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the gastricmucosa of rats exposed to 3.5. h of water immersion restraint stress (WRS) without or withintragastric (i.g.) pretreatment with SIN-1 (5 mg/kg i.g.) or SNAP (5 mg/kg i.g.). Results are mean± SEM of 6-9 rats. Cross (+) indicates significant increase, as compared with the values obtained incontrol group. Asterisk (*) indicates significant decrease, as compared with the respective values inrats exposed to 3.5 h of WRS group.

metabolites, as compared with those in rats exposed to 3.5 h of WRS alone. Thevalue of MDA and 4-HNE mucosal concentration reached minimum in animalstreated with nitroglycerin prior to WRS (Fig. 5).

The MDA results in all investigated groups were significantly higher, ascompared with the respective values obtained in the intact mucosa (Figs 4, 5).

SOD activity

In intact gastric mucosa (control group) SOD activity averaged about347.8±58.0 U/g. Following exposure of rats to WRS, a significant decrease ofSOD activity (245.2±22.0 U/g) was observed. Intragastrical administration ofSIN-1 or SNAP, applied in the doses of 5 mg/kg, resulted in a significant riseof SOD activity, as compared to that observed with WRS (Fig. 6). Similarincrease in SOD activity was observed after pretreatment with NO-ASA (40mg/kg i.g.). Significant increase of SOD activity, as compared with that in WRSmucosa, was demonstrated when nitroglycerin (10 mg/kg i.g.) was used. Afterpretreatment with nitroglycerin, SOD activity reached maximal level(600.3±10.1 U/g) (Fig. 7).

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Fig. 5. Concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the gastricmucosa of rats exposed to 3.5 h of water immersion restraint stress (WRS) without or withintragastric (i.g.) pretreatment with nitroglycerin (10 mg/kg i.g.) or NO-aspirin (NO-ASA 40 mg/kgi.g.). Results are mean ± SEM of 6-8 rats. Cross (+) indicates significant increase, as compared tothe value obtained in the control group. Asterisk (*) indicates significant decrease, as comparedwith the values obtained in group exposed to WRS alone.

DISCUSSION

Nitric oxide (NO) plays an important regulatory role in maintaining gastricmucosal integrity. Previous studies focused mainly on NO as a gastroprotectivefactor, that is released in large quantities in the mucosa and contributes to thereduction of the area and number of gastric lesions. These studies attempted toexplain the mechanisms of NO in cell protection and for this purpose the substrateof NO synthesis (L-arginine) or inhibitors of NO synthase (e.g. L-NNA, L-NAME) were used (16-19). Cooperation between NO and prostaglandins ingastroprotection was described (20). Previous studies using experimental stressmodel, emphasized the importance of NO, released from capsaicin-sensitiveafferent nerves, in the interaction with epidermal growth factor (EGF) on healingof WRS-induced gastric lesions (21). However, little information is availableregarding the participation of reactive oxygen species (ROS) in NO-inducedprotection and accompanying increase of GBF.

Until now, ROS were reported to play a deleterious role in pathomechanism ofgastric mucosal injury. Pohle et al. (22) demonstrated that ROS are involved inaspirin-induced gastric lesions in humans, where treatment with radical

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Fig. 6. Superoxide dismutase (SOD) activity in the gastric mucosa of rats exposed to 3.5. h of waterimmersion restraint stress (WRS) without or with intragastric (i.g.) pretreatment with SIN-1 (5 mg/kg i.g.) or SNAP (5 mg/kg i.g.). Results are mean ± SEM of 6-10 rats. Asterisk (*) indicatesa significant decrease, as compared with the control group. Cross (+) indicates a significant increaseabove the value obtained in WRS alone group.

scavengers prevented the NSAID-induced gastropathy. The ROS mediated lipidperoxidation was proposed by Naito et al. (23) to be a major cause ofindomethacin-induced gastric lesions. In addition, ROS and afferent C-fibershave been proposed to play an important role in the pathomechanisms of thermalstress (24). It was suggested that the ulcerogenesis depends upon the interplaybetween ROS generation and NO action (14). In another study NO wasrecognized as a factor which prevents oxidative stress, for example by theinhibition of leukocyte adherence (25).

Our previous studies documented that ROS are involved in the formation ofgastric mucosal damage due to an enhancing effect on lipid peroxidation andattenuation of mucosal antioxidant mechanisms (15). This notion is in keepingwith our present observations that the production of ROS contribute to thepathomechanisms of stress-induced gastric lesions. This is why we determinedthe effect of NO-donors on ROS generation in rats exposed to 3.5 h of WRS.

Mechanism of protective action of NO-donors appears to be multifactorial.Classic theories may include the vasodilatation, caused by NO-donors, whichultimately leads to the increase in organ perfusion (26-28). Besides gut NO

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Fig. 7. Superoxide dismutase (SOD) activity in the gastric mucosa of rats exposed to 3.5. h of waterimmersion restraint stress (WRS) without or with intragastric (i.g.) pretreatment with nitroglycerin(10 mg/kg i.g.) or NO-aspirin (NO-ASA 40 mg/kg i.g.). Results are mean ± SEM of 6-10 rats. Cross(+) indicates a significant increase, as compared with the value obtained in control group. Asterisk(*) indicates a significant decrease, as compared with the value obtained in control group. Doublecross (++) indicates a significant change as compared with the respective value in rats exposed toWRS alone.

released from SIN-1 exerts anti-atherogenic properties by alteration of LDLmetabolism in macrophages (29). SNAP, another NO donor, strongly influencesthe cardiovascular regulation in hypertension (30). Previous studies revealed thatNO-donors accelerated healing of acute and chronic gastric ulcerations.Nitroglycerin attenuated mucosal damage of ethanol through counteracting effectof this NO-donor on the ethanol-induced fall in potential difference (PD) acrossthe stomach wall (31). The gastroprotective properties of SNAP depend upon theinhibition by this NO-donor of gastric acid secretion that has been documented inisolated parietal cells (32). Moreover SNAP, injected intraperitoneally preventedethanol-induced gastric lesions and this protection was accompanied by increasedgastric blood flow (33).

NO-ASA, despite the inhibition of cyclooxygenase (COX)-activity protectsthe gastric mucosa against damage induced by strong irritants and enhanceshealing of gastric ulcers due to release of NO (34, 35). It was proposed that NO,which is released from NO-ASA, compensates for the deficiency of prostaglandinsynthesis induced by this NSAID (36). Fiorucci et al. (37, 38) demonstrated thatNO-ASA exhibits sparing effect on gastric mucosa by inhibition of apoptosis andattenuation of proinflammatory cytokines such as TNFα and IL-1β. In anotherreport Takeuchi et al. (39) showed protective activity of NO-ASA in a model ofthermic stress. NO-donors can exert an opposite effect after their administrationin high doses. For instance the application of SNAP in high dose delayed healingof ethanol-induced gastric lesions, as demonstrated previously. (33).

So far little information is available regarding the effect of NO-donors on ROSformation in the gastric mucosa. We demonstrated recently that NO-ASA, incontrast to native ASA, failed to delay the healing of chronic gastric ulcer and thiseffect was accompanied by the attenuation of lipid peroxidation (40). Wallace etal. (41) showed that NO-ASA had inhibitory effect on neutrophil adherence to thevascular endothelium. This was attributed to the decrease in neutrophilinfiltration of gastric mucosa, resulting in attenuation of oxidative tissue damage.As mentioned above, NO can also be considered as a pathogenic factorcontributing to tissue damage. Konaka et al. (42) observed intensification of lipidperoxidation and myeloperoxidase activity, during indomethacin-induced smallintestinal lesions in rats, the effect being associated with the increased NOproduction in the intestinal tissue.

Our present study was designed to determine the effect of NO-donors such asSIN-1, SNAP, nitroglycerin, NO-ASA on the lesions induced by WRS. We foundthat NO-donors reduced gastric lesions and that this protection was accompaniedby the fall in oxidative stress parameters, namely decrease of MDA and 4-HNElevels and increase of SOD activity to the level observed in the intact mucosa.The most prominent suppression of lipid peroxidation was observed innitroglycerin-treated gastric mucosa while maximal increase of SOD activityoccurred when SNAP was administered prior to WRS exposure. Thus, weconclude that gastroprotective mechanism of SNAP involves suppression by this

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agent of ROS generation and concomitant increase in SOD activity, that appearsto protect the gastric mucosa from oxidative damage induced by WRS.

In conclusion our present results demonstrated the beneficial role of NO,released from SIN-1, SNAP, nitroglycerin and NO-ASA, in gastroprotectionagainst WRS damage. We also determined that the role of NO-induced protectiondepends upon the attenuation of lipid peroxidation and involves the restoration ofSOD activity in the gastric mucosa subjected to stress.

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R e c e i v e d : October 7, 2002A c c e p t e d : October 29, 2002

Authors address: Prof. S.J. Konturek M.D., Department of Physiology Jagiellonian UniversityMedical School, ul. Grzegórzecka 16, 31-531 Kraków, Poland, tel.: (48-12) 4211006, fax: (48-12) 42311578E-mail: mpkontur@cyf-kr.edu.pl

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