Role of endogenous endothelin-1 in stress-induced gastric mucosal damage and acid secretion in rats

Regulatory Peptides 73 (1998) 43–50

Role of endogenous endothelin-1 in stress-induced gastric mucosal damage and acid secretion in rats Shehta A. Said, Abdalla M. El-Mowafy* Department of Pharmacology and Biochemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt Received 25 April 1997; received in revised form 29 August 1997; accepted 29 September 1997

Abstract In rats subjected to 8 h water-immersion stress, gastric and duodenal mucosal hemorrhage and erosions were detected by macroscopic and histopathological examination. Moreover, plasma and gastric mucosal endothelin-1 (ET-1) levels rose appreciably in a time-related manner during water immersion, with a higher concentration detected in gastric mucosa. Thus, the percentage increases in plasma (gastric mucosal) ET-1, relative to basal levels, after 1, 4 and 8 h of water immersion were 86(172), 169(322) and 210(391)%, respectively. Likewise, a marked increase of gastric acid output was demonstrated 30 min after water immersion and lasted for 3 h. Pretreatment with the endothelin ETA / ET B receptor blocker, bosentan (30 and 100 mg kg 21 ), orally, dose-dependently antagonized gastric and duodenal mucosal damage as indicated by reductions in lesion lengths of 67 and 80%, respectively. Similar protective effects on mucosa were observed when bosentan was given by the intramuscular route. On the other hand, bosentan suppressed the rate of acid output by 30.362.1% in the stressed rats, but had no such effect in non-stressed animals. Taken together, results from this study implicate the endogenous peptide, ET-1, as a powerful mediator of stress-evoked gastro-duodenal mucosal damage and, moreover, present bosentan as a potential protector against hyperacidity and mucosal erosion that occur as a consequence of stress.  1998 Elsevier Science B.V. Keywords: Bosentan; Endothelin level; Gastric acid; Peptic ulcer; Stress; Water immersion

1. Introduction Endothelins (ETs), the 21-amino acid peptides and most potent vasopressor agents, act by stimulating specific cellsurface and G-protein-coupled receptors [1]. Effectors linked to such receptors include phospholipases [2,3], adenylate cyclase [4,5] and protein kinases [6]. Accumulating experimental evidence has implicated ETs in the pathogenesis of several diseases such as bronchospasm [7,8], hypertension [9] and coronary artery diseases [10], diabetes complications [11] and carcinogenesis [12]. On the other hand, elevated plasma ET levels have been observed in numerous stressful conditions such as prolonged exposure to noise [13], birth stress [14], hypovolemic and osmotic stresses [15] and dynamic exercise *Corresponding author. Tel.: 1 20 50 372131; fax: 1 20 50 347900; e-mail: [email protected] 0167-0115 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0167-0115( 97 )01056-2

[16]. Bosentan, a newly-developed competitive ETA / ET B receptor blocker, is a bipyrimidine derivative that devoids intrinsic agonist activity and is among the first generation of the orally-active non-peptide ET receptor blockers [17]. This antagonist has, therefore, been effectively used to counteract the actions of exogenously injected ETs and to verify the involvement of endogenous ETs in various pathophysiological states [9,17–19]. Stress has been a major antecedent in the pathogenesis of peptic ulcer [20], a syndrome that is often life-threatening [21,22]. Local intra-arterial / submucosal injection or systemic infusion of ET-1 induced concentration- and time-dependent gastric mucosal injury and ulceration [23– 25]. Similarly, in an isolated and vascularly perfused rabbit stomach model, ethanol-induced gastric ulceration correlated well with its ability to release ET-1 in the perfusate [26]. However, there is a paucity of information regarding the role of endogenous ETs in the pathogenesis of stress-

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induced gastric mucosal erosions. Likewise, a controversy revolves around the effects of ETs on gastric acid secretion. Thus, i.v. injection of ETs reduced gastric acid secretion in rats [27]. On the other hand, ET-1 was found to activate ‘maxi’ Cl 2 channels in guinea pig gastric parietal cells, implying a contribution in gastric acid secretion [28,29]. Accordingly, the objectives of this study were to seek the potential release and contribution of ET-1 in stress-induced acid secretion and gastrointestinal mucosal damage.

2. Materials and methods

2.1. Materials 2.1.1. Animals used Male Sprague–Dawley rats weighing 200–250 g were used in this study 2.1.2. Chemicals Bosentan, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2methoxy-phenoxy)-2,29-bipyrimidin-4-yl]benzenesulphonamide monohydrate, was kindly provided by F. Hoffmann La Roche Ltd., Basel, Switzerland, and was prepared as an aqueous suspension in 0.5% carboxymethylcellulose (CMC). Endothelin assay kit (ELISA) was purchased from Amersham Corporation (Arlington Heights, IL, USA). Aprotinin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were of analytical grade.

3. Methods

3.1. Water immersion stress-induced mucosal damage Thirty-six male rats were divided into six equal groups. Rats were deprived of food 24 h prior to the experiment. Two groups received bosentan suspension in doses of 30 and 100 mg kg 21 orally and another two groups received these same doses of bosentan by intramuscular (i.m.) injection. The last two groups received the vehicle only by oral or i.m. routes. One hour after bosentan (vehicle) administration, rats were subjected to water-immersion stress as described by Kitagawa et al. [30]. Briefly, animals were lightly anesthetized with ether, restrained on a wooden plate and immersed vertically in water to the level of xiphoid process in a water bath thermostatically controlled at 23618C, for 8 h. Rats were killed by decapitation and stomachs and duodena were removed and opened along the lesser curvature of the stomach. Photographs of the stomach and duodenum were taken and the extent of damage visible macroscopically, mainly hemorrhagic and

erosive lesions, was measured as the sum of the length (mm) of the elongated or punctated lesions in each stomach. Also, the percentage of the eroded mucosal area in each stomach was recorded. Samples from these organs were excised, dehydrated, embedded in paraffin, sectioned at 6 mM, stained with hematoxylin and eosin and examined under a light microscope. Microscopic estimation of gastric mucosal injury was made using the criteria of Whittle et al. [32]. Briefly, a 1 cm histological section was assessed for epithelial cell damage, glandular disruption or vasocongestion in upper mucosa, hemorrhage in the mid to lower mucosa, and deep necrosis or ulceration. The number of animals showing these histopathological lesions in each group was recorded and compared with that of the corresponding control group.

3.2. Gastric acid output in stressed and non-stressed animals The method described by Kitagawa et al. [31] was adopted to study acid output in stressed animals. Thus, 12 male rats were deprived of food for 16 h, but allowed drinking water. Animals were divided into two equal groups, lightly anesthetized with ether and restrained by tying their legs onto a wooden board. For each animal, a transverse incision was made in the abdomen. An open polyethylene cannula was placed in the forestomach via an incision in the duodenum and ligated about 0.5 cm from the pylorus. The esophagus was ligated near the stomach. The incisions were closed with adhesive, and ether was discontinued. To remove any solid contents, the stomach was washed with 2 ml of saline at 378C three times, at 5 min intervals. Two milliliters of normal saline warmed to 378C was placed in the stomach, left for 30 min and then aspirated and replaced by a fresh solution. The process was repeated twice to obtain acid secretion before water immersion. Animals of the first group received bosentan (100 mg kg 21 i.m.) while those of the second group were injected with an equivalent volume of the vehicle (2.5 ml kg 21 of 0.5% CMC). All rats were thereafter immersed vertically in water at 2360.58C to the depth of the xiphoid and gastric fluid samples were collected every 30 min for 3 h after bosentan (vehicle) treatment. The aspirated fluids were titrated to pH 7.0 with 0.01 N NaOH, using a pH meter, and acid output was calculated as mEq / 30 min. The effect of bosentan on acid secretion in non-stressed animals was examined using two groups of male rats (six each). Animals were anesthetized with urethane (1.4 g kg 21 , i.p.) and prepared for determination of gastric acid as mentioned above. One group received bosentan (100 mg kg 21 i.m.), while the other was injected with the vehicle. Acid output was assessed exactly as for stressed animals.

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3.3. Measurement of plasma and gastric mucosal ET-1 level Rats, six animals per group, were subjected to waterimmersion stress for 0, 1, 4 and 8 h, before they were decapitated. Blood was collected into tubes containing 7.5 mM EDTA. The protease inhibitor, aprotinin (500 KIU ml 21 ), was also added. Plasma was separated immediately by centrifugation at 2000g for 10 min at 48C and then kept at 2 158C prior to assay. For gastric mucosal samples, after abdominal incision, the forestomach was cut and the superficial mucosa of the glandular stomach was cut with small scissors. ET-1 in plasma or mucosa was then extracted. Briefly, 1 ml of plasma or

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0.2 g of mucosa were treated with 0.25 and 1.0 ml 2 M HCl, respectively, and the mucosal slurry was then homogenized. All tubes were centrifuged at 1000g for 5 min. Supernatants were loaded to Amersham’s AmPrep TM , 500 mg C2 columns. Columns were then washed with 5 ml H 2 O 1 0.1% trifluoroacetic acid (TFA) and ET-1 was eluted with 2 ml of 80% methanol / H 2 O 1 0.1% TFA. Solvent was removed under nitrogen and the pellet reconstituted in 250 ml of assay buffer. ET-1 levels were determined by sandwich-ELISA assay using a kit from Amersham. The assay endpoint is a peroxidase / diaminobenzidinebased colorimetric determination and the range of standard ET-1 used was 1–16 fmol(2.49–79.75 pg) / well.

Fig. 1. Photographs of stomach (S) and duodenum (D) 8 h after water-immersion stress, showing severe ulceration and hemorrhage in control (A), and minimal lesions in bosentan-treated (B) rats.

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4. Statistical analysis The mean lesion length in bosentan-treated and control groups was compared using Student’s ‘t’ test. The incidence of various pathological lesions in gastric mucosa in bosentan-given groups was compared with that of the control using the x 2 test.

5. Results

5.1. Effect of bosentan on water-immersion stressinduced mucosal damage Macroscopic examination of the stomachs and duodena

indicated that water-immersion stress caused severe gastrointestinal mucosal damage and hemorrhage, especially in the glandular part of the stomach (Fig. 1). An average mucosal lesion area of 8566% and 5464% was found in the stomachs and the proximal duodena of the control groups, respectively. The glandular stomach showed punctated and elongated lesions with a mean lesion length of 36.863.4 and 34.363.1 mm in the control groups that had received the vehicle by oral and i.m. routes, respectively (Fig. 2). Bosentan, given by oral (intramuscular) routes, inhibited gastric injury dose-dependently. Thus, the areas of damaged mucosa were 1262 (10.461.6) and 861.3 (9.161.6)% of the total mucosal area following treatment with 30 and 100 mg kg 21 of bosentan, respectively. A corresponding reduction in lesion length of 67(65) and 80(78)%, respectively, was obtained (Fig. 2). The incidence of duodenal lesions was, likewise, effectively reduced by bosentan. Average reductions in lesion length by 30 and 100 mg kg 21 bosentan were 5166 and 6865% (data not shown). Microscopic examination indicated the presence of epithelial cell damage, glandular disruption, mucosal hemorrhage and ulceration (necrosis) in the stomachs of all rats in the control group. Although the incidence of epithelial cell damage was not significantly reduced in bosentan-treated groups, those of mucosal hemorrhage and damage were markedly decreased (P , 0.05). Results for the ability of oral and i.m. bosentan to antagonize stressinduced gastric lesions virtually matched (Table 1).

5.2. Effect of bosentan on gastric acid secretion in stressed and non-stressed rats

Fig. 2. Inhibitory effect of oral (p.o.) and intramuscular (i.m.) bosentan treatment on gastric lesions produced by water-immersion stress in rats. *Significant difference from the corresponding control group (P , 0.05) using Student’s ‘t’ test.

As can be seen in Fig. 3A, immersion stress virtually doubled the rate of gastric acid secretion in control group after 30 min of water immersion (P , 0.05). The increased rate of gastric acid secretion in the vehicle-treated group maintained almost a constant level for up to 3 h following water immersion. Bosentan treatment significantly attenuated the stress-induced rise in acid secretion. Average

Table 1 Effect of bosentan on water-immersion stress-induced mucosal damage Treatment

No. of animals showing (out of six) Epithelial cell damage

Vehicle (p.o.) (i.m) Bosentan 30 mg kg 21 (p.o.) (i.m.) 100 mg kg 21 (p.o.) (i.m.)

Glandular disruption

Midmucosal hemorrhage

Ulcerations or necrosis

6 6

6 6

6 6

6 6

5 6

2 3

1* 2

1* 1*

3 4

1* 1*

1* 0*

0* 1*

*Significantly different from control groups (P , 0.05) using the x 2 test.

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immersion were, respectively, 86, 169 and 210% above the basal level. Gastric mucosal ET-1 increased at a much higher rate of 172, 322 and 391%, respectively. Bosentan treatment had no significant effect on the elevated ET-1 levels (data not shown).

6. Discussion

Fig. 3. Effect of bosentan (100 mg kg 21 , i.m.) on acid secretion rate of water-immersion stressed (A) and non-stressed (B) rats. * Significantly different from vehicle-treated group (P , 0.05).

reduction in total rate of acid output during water immersion was 30.362.1%. Similar treatments with bosentan in non-stressed animals (Fig. 3B) showed no significant difference in acid secretion between bosentan- and vehiclegiven groups.

5.3. Plasma and gastric mucosal ET-1 levels Basal ET-1 levels were 1.4560.21 pg / ml plasma and 35.664.5 pg / g gastric tissue. These levels were appreciably increased after 1 h of water immersion and continued to rise markedly with stress time (Fig. 4). The increments in the plasma ET-1 level after 1, 4 and 8 h of water

Water-immersion stress has been introduced as a model for acute induction of peptic ulcer [31]. Elevated plasma ET levels have been observed in numerous stressful conditions [12–15]. This, however, is not the case with some kinds of stress such as that evoked by food deprivation, wherein plasma ET-1 was strikingly reduced [33]. The possible contribution of ET-1 to water-immersion stress-induced gastric ulcer and its prevention are currently being investigated in rats. Three- and five-fold rises in plasma and gastric mucosal ET-1, respectively, were observed in this study following water-immersion stress and correlated well with the severity of the produced ulceration. Several mediators have been implicated in the pathogenesis of gastric mucosal damage. These include lipid peroxides and free-radicals [34,35], neutrophil adherence [36], platelet activating factor (PAF) [37] and thromboxane-A 2 (TXA 2 ) overproduction [30,38]. The mechanism whereby ETs trigger their ulcerogenic effects is not fully understood. It is well documented that ETs enhance the production of free radicals, superoxides, from human neutrophil and rat lung [39,40], promote the action of PAF [37] and induce TXA 2 production [41]. Presumably, these findings indicate a role by such mediators in ET-1-induced gastric erosion. This hypothesis may be argued against, at least in part, by the finding that indomethacin, in sub-ulcerogenic but cyclo-oxygenase inhibiting doses, and the lipoxygenase inhibitor BWA4C, had no effect on ET-1-induced gastric damage [23]. Likewise, ET-1’s ulcerogenic effects were affected neither by OKY1581, a thromboxane synthase inhibitor, nor by an anti-neutrophil serum, but were partially antagonized by the PAF receptor blocker WEB2086 [42]. Nonetheless, it remains unequivocal that perturbation of the gastric microcirculation ending with ischemia is the major predisposing factor to gastric mucosal ulceration [43]. ET-1, on a molar basis, is the most powerful vasopressor ever isolated [44]. In the current study, higher ET levels were detected in gastric mucosa than in plasma, implying that the ulcerogenic ET-1 mostly originates from the mucosal vascular endothelium to exert its ischemic effects in a paracrine, rather than an endocrine, manner. Similar observations on the mode of ET action have been reported [45,46]. In eliciting their actions in mammalian tissues, ETs activate two major subtypes of receptors: namely, the ETA and ET B receptors [2,6]. The ET-1-induced rat gastric

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Fig. 4. Time course for the water-immersion-induced increase in rat plasma and gastric mucosal ET-1 levels.

mucosal damage was ascribed to ETA -dependent vasoconstriction, based on attenuation of the ET-1 effects following pretreatment with the ETA receptor antagonist, BQ123. This antagonist was effective in reducing hemorrhage in midmucosa but not the glandular disruption or epithelial cell damage [45]. In this study, the use of the mixed ETA / ET B antagonist, bosentan, dose-dependently reduced the severity of the mucosal damage and partly protected against glandular disruption and epithelial damage at an oral or intramuscular dose of 100 mg kg 21 . Thus, although implicating ET B receptors in the current study seems a premature assumption in light of the different models used, these studies show bosentan as a potent protector against stress-induced gastric mucosal damage. Data obtained on the protective ability of bosentan given by oral and i.m. routes were highly matched. This, unequivocally, eliminates the possibility of local protective effects on gastric mucosa by the orally-given ET-receptor blocker, bosentan. For several years following the discovery of ET-1, it was believed that ETA receptors transduced the vasopressor effects, while ET B receptors, via nitric oxide, mediated the vasodilatory actions of endothelins [6,46]. Recent evidence, however, indicates that ET B receptor subtypes may constrict various vascular beds in rats and humans [47,48]. On the other hand, besides its possible ischemic effects in rat gastric microcirculation, ET B receptor is the likely inflammatory candidate releasing nitric oxide and vasodilatory prostaglandins in rat vascular beds in response to lower ET-1 concentrations, thereby mediating hyperemia, gastric albumin extravasation and endothelial

injury that occur following gastric intra-arterial injection of ET-1 [49]. The influence of secretagogues and stress on gastric acid secretion has been widely studied. Many investigators reported a parallel increase in mucosal blood flow (MBF) and acid secretion when animals are given various secretagogues, thereby maintaining the MBF-to-acid output ratio and protecting the gastric mucosa against acid injury [50,51]. Conversely, in stressful situations such as that induced by water immersion, acid output increases immediately and progressively, with the MBF remaining unaffected, resulting in a lowering of the MBF / acid output ratio and, consequently, increasing the incidence of mucosal erosion and ulceration [30]. The impact of ETs on gastric acid is uncertain. Studies carried out on guinea pig gastric parietal cells showed ETs to activate ‘maxi’ Cl 2 channels via ET B receptors, implying a contribution by these receptors to gastric acid secretion [28,29]. On the contrary, in rats, i.v. injection of ET-1 or ET-3 reduced gastric acid secretion [27], and the suppression of gastric acid output following portal hypertension has been ascribed to local release of ET-1 [52]. In this study and others [30], water-immersion stress significantly increased gastric acid output. Although the role of ETs in acid secretion is controversial, its robust and maintained reduction of MBF is well established [34,49], an effect that would substantiate the deleterious response to gastric acid secreted during stress by other known mediators. However, in this study, bosentan reduced the rate of acid output in stressed rats by 30%, but had no effect in non-stressed

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animals that had been urethane-anesthetized. In agreement with several studies, such anesthesia was inevitable to assess acid secretion under non-stressing conditions. Furthermore, unlike bosentan in this study, the antisecretory agents cimetidine, ranitidine and nazitidine were repeatedly shown to suppress up to 80% of acid secretion in anesthetized rats [53,54]. Therefore, the failure of bosentan to alter acid secretion in non-stressed animals excludes the possibility of actions on non-ET systems that are involved in acid secretion such as the cholinergic and histaminergic systems. Furthermore, this may imply that ET-1 is more involved in the pathogenesis of stress-induced ulcer than in physiological regulations of gastric functions. Bosentaninduced acid reduction is an essential participant in the mechanism whereby bosentan abates mucosal damage. Other mechanisms, such as improving MBF, cannot be ruled out but remain, however, to be delineated. In conclusion, the present study suggests a role for endogenous ET-1 in the etiology of stress-evoked peptic ulcer and presents the mixed ET receptor blocker, bosentan, as a potent protector against such complication.

[9]

[10]

[11]

[12]

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[16]

Acknowledgements We wish to thank Dr. Ahmed Said, Department of Pathology, School of Medicine, Mansoura University, for his assistance with the histopathological examinations. We are very grateful to Dr. Martin Clozel and Dr. Eva-Maria Gutknecht, Hoffmann La Roche, Ltd., Basel, Switzerland, for providing us with the bosentan.

[17]

[18]

[19]

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