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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
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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.
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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
764
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.
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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
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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
766
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.
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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.
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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.
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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
769
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.
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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
770
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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: [email protected]
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