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Protective role of nitric oxide in indomethacin-induced gastric ulceration by a mechanism independent of gastric acid secretion

Pharmacological Research, Vol. 43, No. 5, 2001doi:10.1006/phrs.2001.0801, available online at on PROTECTIVE ROLE OF NITRIC OXIDE IN INDOMETHACIN-INDUCED
MAHMOUD M. KHATTABa, MOHAMED Z. GADb,∗ and DALAAL ABDALLAHa aPharmacology Department, Faculty of Pharmacy, Cairo University, Egypt, bBiochemistry Department, Faculty of Pharmacy, Cairo University, Egypt Gastric ulceration was induced in rats by i.p. injection of the non-steroidal anti-inflammatorydrug (NSAID), indomethacin (IND) (30 mg kg−1). Pyloric ligation was carried out in each animalbefore injection to enable collection of the gastric juice. Three hours later, the animals were killedand their stomachs were removed. In the gastric juice, the amounts of mucin, pepsin and HCl wereassessed. Gastric mucosa were scrapped for the determination of nitric oxide (NO) (as nitrite) afterevaluation of the gastric ulcer index.
The influence of arginine (ARG) (300 mg kg−1), a NO precursor, NG-nitro-L-arginine methyl ester (L-NAME) (50 mg kg−1), a non-selective constitutive nitric oxide synthase/inducible nitricoxide synthase (cNOS/iNOS) inhibitor, and the selective iNOS inhibitor aminoguanidine (AMG)(50 mg kg−1) were studied. Each NO modulator was injected i.p. 30 min before IND administration.
Results indicated that IND elevated gastric acidity by 80% of the normal group, decreased non-significantly mucosal nitrite by 22% and exhibited a remarkably high ulcer index (χ = 17). Neithermucin nor pepsin levels were significantly altered. In comparison with the IND group, pretreatmentwith L-NAME caused a significant decrease in gastric HCl, further decrease in mucosal nitrite (50%of normal) and a two-fold increase in the ulcer index score (χ = 34), despite the decrease in HCl.
AMG did not alter gastric acidity, decreased mucosal nitrite by 38% of the normal value and failedto alter significantly the ulcer index of IND. On the other hand, pretreatment with ARG did not alterthe gastric acidity and raised mucosal nitrite by 10% above normal. Surprisingly, ARG improvedthe gastric ulcer score (χ = 1) almost similar to the normal score (χ = zero). Therefore, this studycreates a new pathway for the potential treatment of NSAID gastric ulceration through modulationof NO synthesis, regardless of the effect on gastric acidity.
KEY WORDS: nitric oxide, indomethacin, gastric ulcer, arginine, NOS inhibitors.
induced by various agents However, the role ofNO in regulation and maintenance of the functions of The use of non-steroidal anti-inflammatory drugs gastric mucosa has not yet been fully understood.
(NSAIDs) is limited by their ulcerogenic activity as The present investigation was therefore designed to well as their ability to interfere with ulcer healing.
explore the biochemical effects of the NO precursor, These effects are mainly mediated through inhibition of L-arginine (ARG), and the nitric oxide synthase (NOS) prostaglandin synthesis Recently, many strategies inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), have been adopted to minimize the NSAID-induced gastropathy. These include the development of NSAIDs (cNOS/iNOS) inhibitor, and the selective iNOS inhibitor that only inhibit the inducible isoform of cyclooxygenase aminoguanidine (AMG) on indomethacin (IND)-induced (COX-2) [] and coupling of NSAIDs with a nitric oxide gastric ulceration, gastric acid secretion as well as mucin (NO)-releasing molecule NO donors have been re- and pepsin secretions. The level of the mucosal NO was peatedly shown to protect gastric mucosa against damage examined after each treatment. The ultimate goal wasto understand the role of NO in the regulation of gastric ∗Corresponding author. Associate Professor, Biochemistry Department, acid secretion and to search for a simple protective Faculty of Pharmacy, Cairo University, Kasr El-Aini st, Cairo 11672, mechanism against NSAID-induced gastropathy.
Pharmacological Research, Vol. 43, No. 5, 2001 MATERIALS AND METHODS
20 min was measured colorimetrically at 680 nm. A blankexperiment was similarly carried out using 1 ml of N/100 HCl. In addition, a standard solution containing 0.2 ml Male Wistar albino rats weighing 150–200 g were used working tyrosine standard made up to 1 ml with N/100 in this study. The rats were treated in humane conditions and were allowed free access to water and diet ad libitumuntil the beginning of the experiment. All experimental protocols were approved by the Animal Care Committee The method adopted is based on the determination of the hexose component of the mucin. It depends on thereaction of carbohydrate in concentrated sulfuric acid with Orcinol (5-methyl resorcinol) to give a colored Rats were deprived of food, but not water, for about product that can be measured colorimetrically. In detail, 18–24 h before the experiment. For each rat, the abdomen 0.25 ml of 1 : 20 diluted juice was added to an equal was then incised and the pylorus was ligated under ether volume of 1.6% Orcinol and 2 ml of 60% sulfuric acid.
anesthesia. Indomethacin (30 mg kg−1 suspended in 1% The mixture was boiled in a water bath for 10 min and Tween 80) was given intraperitoneally immediately after then cooled on ice. The optical density was measured at pyloric ligation. Three hours later, animals were killed 425 nm. Results are expressed as mg hexose dl−1 with an ether overdose. A normal group that was treatedsimilarly but without administration of indomethacin was Determination of gastric ulcer index After collection of the gastric juice, the stomach of each animal was then rinsed with saline and inflated on cork.
The ulcer index was expressed as the sum of the length Three groups of rats were given intraperitoneally (mm) of all lesions in the fundic region [].
30 min before pyloric ligation either the NO precursor, L-arginine (300 mg kg−1), or the NOS inhibitors L-NAME Determination of mucosal nitric oxide (50 mg kg−1) or AMG (50 mg kg−1). Doses of drugs Finally, the gastric mucosa of each rat was scrubbed were in homogeny with previous reports , ].
off, weighed and its nitric oxide content was determinedas nitrite by diazotization with sulfanilic acid at acidic pH and subsequent coupling with N-1-naphthyl-ethylene After the animals were killed, their stomachs were re- diamine to give a colored product that was measured moved following ligature of the oesophocardiac junction, washed with distilled water and dried between filter paperand opened along the greater curvature. The gastric juice was drained and centrifuged at 3500 rpm and used for Data are presented as mean ± SEM for 6–10 animals.
Statistical significance between groups was evaluatedusing analysis of variance (ANOVA). In all data analysis, P values of 0.05 or less were considered significant Titratable acidity in the supernatant of the gastric juice was determined by titration with 0.01 M NaOHusing phenol red as an indicator. Units are expressed as As shown in Table IND elevated significantly gastric acidity by 80% above that of the normal group. This Peptic activity is a major factor involved in the prote- elevation was normalized by pretreatment with L-NAME.
olytic activity of gastric secretion. Measured pepsin ac- Pretreatment with AMG or ARG failed to significantly tivity represents the amount of liberated tyrosine (µmol) modify the elevated gastric acid secretion. Neither IND per 1 ml of gastric juice per minute, using 1:100 diluted nor any pretreatment had a significant effect on mucin gastric juice and 2% bovine albumin in N/100 HCl as a content or peptic activity of the gastric juice.
substrate. In detail, each sample of the gastric juice was With regard to the effects on gastric ulcer formation, first diluted 1:100 with N/100 HCl. One ml of the diluted IND treatment induced a remarkably high ulcer index juice was added to 5 ml of 2% bovine serum albumin so- (x = 17) (Fig. ). Pretreatment with L-NAME further lution. The mixture was incubated at 37 ◦C for exactly aggravated the ulcer formation as reflected by a two- 10 min in a water bath. At the end of the incubation pe- fold increase in the ulcer index score (x = 34). AMG riod, 10 ml of 0.3 M trichloroacetic acid was then added pretreatment failed to alter significantly the ulcer index and the mixture was boiled for 5 min. The solution was score as compared to the IND index. On the other hand, then centrifuged for 5 min at 3000 rpm and filtered. To ARG profoundly improved the gastric ulcer score (x = 1) 1 ml of the filtrate 2 ml of 0.5 N NaOH and 0.2 ml of to almost that of the normal score (x = 0).
Folin reagent were added. The color that developed after Concerning the effect of treatments on mucosal nitrite Pharmacological Research, Vol. 43, No. 5, 2001 HCl level, mucin content and pepsin activity in the gastric juice of normal, indomethacin, L-NAME, aminoguanidine, and L-arginine treated
rats. Results are mean ± SEM of 6–10 animals
a Significantly different from IND at P< 0.01. bSignificantly different from normal at P< 0.05. Units are mEq l−1 for HCl, mg hexose dl−1 for mucin, µmol ml−1 min−1 for pepsin.
with the NO precursor, L-arginine, and the aggravation of ulcer formation by pretreatment with the NOS inhibitor, L-NAME. These effects were, interestingly, independent of gastric acid secretion or other measured factors such as pepsin activity or mucin content. Rather, they are well correlated with mucosal nitrite level.
In support of this conclusion, several reports from dif- ferent laboratories have demonstrated the importance of endogenous NO in the protection of gastric mucosa. Two studies from Pique’s laboratory [, ] have shown thatNO plays a vasodilatory role in the gastric microcircula- Ulcer index (mm) of normal, indomethacin, L-NAME, tion during acid secretion. Other studies have accredited aminoguanidine, and L-arginine treated rats. Results are mean ± SEMof 6–10 animals. (∗∗) Significantly different from indomethacin at P< the role of NO as an endogenous modulator of leukocyte adhesion In support, Calatayud et al. [haverecently shown that transdermal nitroglycerin protectedagainst indomethacin-induced gastric ulceration through maintenance of mucosal blood flow and reduction of leukocyte–endothelial cell rolling and adherence. In ad- dition, Wallace ] stated that reduction of gastric blood flow is the main predisposing factor in the induction of Reports have not restricted the role of NO to gastric protection, but also include the acceleration of ulcer healing. Konturek et al. [have shown that glyceryltrinitrate was capable of ulcer healing and suppression of NO synthesis resulted in impaired ulcer healing. It is possible that NO directly accelerates ulcer repair by Level of mucosal nitrite (µg g−1 tissue) in normal, in- promoting the growth of smooth muscles as suggested by domethacin, L-NAME, aminoguanidine, and L-arginine treated rats. Re- sults are mean ± SEM of 6–10 animals. (∗) Significantly different from Our data show a close agreement between the preven- indomethacin at P< 0.05. (++) Significantly different from normal at tion of ulcer formation produced by L-arginine against indomethacin-induced ulceration and the maintenance ofmucosal NO. This agreement was also found for the ef- (Fig. IND decreased non-significantly mucosal nitrite fect of the NOS inhibitor, L-NAME, which aggravated ul- by 22%. In contrast, all pretreatments, as anticipated, cer formation. It is noticeable that, although AMG, a se- affected mucosal nitrite content either by reduction (50% lective iNOS inhibitor, lowered significantly gastric mu- with L-NAME and 38% with AMG) or elevation (10% cosal nitrite, it failed to significantly alter indomethacin- with ARG) as compared with the values of the normal induced damage. This suggests that cNOS may be the ma- Other than the role of NO in maintenance of blood flow, NO may protect against NSAID damage by pro- DISCUSSION
motion of prostaglandin synthesis. A mutual interactionwas shown to exist between NOS and cyclooxygenase The present study refers to the importance of mucosal NO (COX) enzymes. NO donors were shown to enhance in the protection against NSAID-induced gastric ulcera- COX activity whereas NOS inhibitors blocked PGE2 tion. Evidence is clearly demonstrated by the prevention production In addition, indomethacin, a COX of IND-induced gastric ulcer formation by pretreatment inhibitor, reduced the level of cyclic GMP that was Pharmacological Research, Vol. 43, No. 5, 2001 increased by NO donors [This may explain the 22% decrease in mucosal NO by indomethacin administration 2. Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, Isakson PC, Seibert K. Selective inhibition of in-ducible cyclooxygenase 2 in vivo is anti-inflammatory and nonul- Results of the studies investigating the effect of NOS cerogenic. Proc Natl Acad Sci USA 1994; 91: 3228–32.
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