European Journal of Pharmacology, 211 (1992) 277-279
277
© 1992 Elsevier Science Pubhshers B V. All rights reserved 0014-2999/92/$05.00
EJP 21000
Short communication
Role of nitric oxide and prostaglandins in sucralfate-induced gastroprotection Stanislaw J. K o n t u r e k , T o m a s z Brzozowski, J o l a n t a M a j k a a n d K r z y s z t o f Czarnobilski Instttute of Phystology, UnwersttyMedtcal School, Krakow, Poland Recewed 26 November 1991, accepted 10 December 1991
We investigated the role of nitric oxide (NO) and prostaglandins (PG) in the prevention by sucralfate of ethanol-induced gastric damage and the decrease of gastric blood flow and compared them with those obtained with nocloprost, a potent locally acting gastroprotective agent. Sucralfate and nocloprost given mtragastrically (i.g.) protected dose dependently the gastric mucosa against the damage by absolute ethanol and prevented the decrease in blood flow induced by ethanol. Pretreatment with NG-nltro-L-arglnine (L-NNA), an inhibitor of NO synthase decreased dose dependently the protection and the maintenance of blood flow provided by sucralfate but not by nocloprost. Th~s decrease of sucralfate protection was antagonized by L-arginine but not D-argmine. Pretreatment with indomethacin also reversed, in part, the protective and hyperemic effects of sucralfate but the combination of both indomethacln and L-NNA completely abolished these effects. We conclude that sucralfate activates both the NO and PG systems that cooperate in the gastroprotective action of this drug and that NO is not involved in the protection induced by a PGE: analog. Nitric oxide (NO); Gastroprotection; Blood flow (mucosal); Nocloprost
I. Introduction
2. Material and methods
Sucralfate, a complex of sucrose octasulfate and aluminium has been shown in numerous studies to prevent the formation of acute gastric mucosal lesions induced by various ulcerogens (Konturek et al., 1987), and this has been explained, in part, by the stimulation of prostaglandin (PG) biosynthesis by this drug. Recently, the increase in mucosal blood flow accompanying the gastroprotective action of capsaicin has been attributed to the biological activity of endothelium-derived relaxing factor, nitric oxide (NO), which was proposed to interact with mucosal prostanoids on both gastric blood flow and tissue integrity (Whittle et al., 1990; Peskar et al., 1991). We have, therefore, investigated the possible role of NO and PG in gastroprotection and gastric blood flow induced by sucralfate and compared their effect with that of nocloprost, a locally active gastroprotective and vasoactive agent (Konturek et al., 1991; Hui et al., 1991). For this purpose we used the inhibitor of NO biosynthesis, NG-nitro-L-arginine (L-NNA), and the substrate for NO synthase, Larginine (Sigma Chemicals Co., St. Louis, MO, U.S.A.).
Groups of 8-10 male Wistar rats (160-180 g) received L-NNA (12.5-50 m g / k g i.v.) a n d / o r indomethacin (5 m g / k g i.p.) or vehicle (saline, 1 ml/kg). Additional groups of rats received either L-arginine or D-arginine (100 m g / k g i.v. or the vehicle (1.0 m l / k g ) 15 min before L-NNA. About 15 min later sucralfate administered orally in a dose (100 m g / k g ) that we had found to induce almost complete gastroprotection against absolute ethanol (Konturek et al., 1987). In control experiments, 1 m l / k g of vehicle was used 15 min after pretreatment with L-NNA, or the combination of L-arginine or D-arginine plus L-NNA. In tests of the pretreatment with indomethacin, L-NNA (50 m g / k g i.v.) or sucralfate (100 m g / k g ) was administered and 100% ethanol was applied 30 min later. For comparison, a stable P G E 2 analog, nocloprost, was used instead of sucralfate at a dose (1 /zg/kg i.g. (intragastrically)) that was shown to induce almost complete gastroprotection against ethanol. A further 15 min after the application of sucralfate or nocloprost, 1.5 ml of 100% ethanol was instilled into the stomach. The rats were anesthetized 60 min later with urethane sodium pentobarbitone (60 m g / k g i.p); the abdomen was exposed by a midline incision and a small incision was made in the forestomach to allow emptying of
Correspondence to' S.J Konturek, Institute of Physiology, ul. Grzegorzecka 16, 31-531 Krakow, Poland
278 TABLE 1 Effects of L-NNA (50 mg/kg iv), indomethacm (5 mg/kg i.p), sucralfate (100 mg/kg Lg.) and nocloprost (1 /zg/kg i.g) with or without addition of L-argmme or D-arglmne (100 mg/kg) on the area of ethanol-reduced gastric lesion and gastric blood flow m rats Means+_S E.M. of 10-20 rats Lesion area
(mm2)
Blood flow (ml/min per 100 g)
Vehicle + ethanol (control)
88_+ 6
L-NNA+ ethanol
121 + 18 a
17+_3 a
17+_ 3 a
45+_7 a
68 +_ 7 b
22 +_4 b
20 +_ 4 a
37 +_5 a
76 5 : 8 a
20 +_3 b
94 +_19
17 +_3
38+ 4 b
31+5 b
Sucralfate+ethanol L-NNA + sucralfate + ethanol L-Arg + L-NNA + sucralfate + ethanol D-Arg + L-NNA + sucralfate + ethanol Indomethacm + vehicle + ethanol Indomethacln + sucralfate+ethanol Indomethacm + L-NNA +ethanol Indomethacm + L-NNA + sucralfate + ethanol Nocloprost +ethanol L-NNA + nocloprost + ethanol
23 +-2
127_+ 18 a
16+3
117_+27 b
19-+4 b
21 +_ 4 a
52+_7 a
28+_ 4 a
40+_5 a
a
a Slgmflcant (P < 0.05) change as compared to vehicle control values. b Sigmficant changes as compared with sucralfate + ethanol.
gastric c o n t e n t to t h e e x t e r i o r a n d for r e c o r d i n g o f gastric b l o o d flow using a laser D o p p l e r f l o w - m e t e r ( M o d e l B P M 403A, V a s a m e d i c s Inc., St. Paul, M N , USA). Finally, the s t o m a c h was r e m o v e d , o p e n e d a n d the a r e a o f gastric lesions was d e t e r m i n e d by p l a n i m e t r y . M e a n s ___S.E.M. w e r e c a l c u l a t e d for t h e lesion a r e a and m u c o s a l b l o o d flow. Statistical significance w e r e e v a l u a t e d using W i l c o x o n ' s r a n k test.
r e d u c t i o n in gastric b l o o d flow a n d t h e p r e t r e a t m e n t with L - N N A r e s u l t e d in a f u r t h e r significant d e c r e a s e o f this flow. L - A r g i n i n e (100 m g / k g ) b u t n o t D - a r g i n i n e given b e f o r e L - N N A (50 m g / k g ) p a r t l y r e s t o r e d the b l o o d flow r e d u c e d by t h e c o m b i n a t i o n o f L - N N A + e t h a n o l (table 1). P r e t r e a t m e n t with i n d o m e t h a c i n d i d n o t affect significantly t h e e t h a n o l - i n d u c e d gastric lesion a r e a or the b l o o d flow b u t r e v e r s e d in p a r t t h e p r o t e c t i v e effects o f sucralfate. T h e c o m b i n a t i o n o f i n d o m e t h a c i n plus LNNA completely abolished the mucosal protection and t h e rise in t h e b l o o d flow c a u s e d by sucralfate. A d m i n i s t r a t i o n o f n o c l o p r o s t (1 / x g / k g i.g.) gave a l m o s t c o m p l e t e p r e v e n t i o n of e t h a n o l - i n d u c e d d a m age a n d c h a n g e s in gastric b l o o d flow. P r e t r e a t m e n t with L - N N A (50 m g / k g ) o r the c o m b i n a t i o n of La r g i n i n e + L - N N A d i d not affect significantly t h e gast r o p r o t e c t i o n or m u c o s a l circulation a f t e r nocloprost.
4. Discussion T h e p r e s e n t results d e m o n s t r a t e indirectly, using a p o t e n t N O synthase i n h i b i t o r ( L - N N A ) , t h a t N O is involved in s u c r a l f a t e - i n d u c e d g a s t r o p r o t e c t i o n a n d suggest t h a t this involvement is r e s p o n s i b l e for the i n c r e a s e in m u c o s a l circulation. Since sucralfate is not a d s o r b e d from t h e g a s t r o i n t e s t i n a l l u m e n it r e m a i n s to be d e t e r m i n e d w h a t is t h e m e c h a n i s m o f activation o f t h e N O system by this drug. N o c l o p r o s t p r o t e c t e d the m u c o s a even a f t e r t h e b l o c k a d e o f N O synthase by L - N N A , i n d i c a t i n g t h a t N O is not involved in P G - i n d u c e d g a s t r o p r o t e c t i o n . Since t h e p r e t r e a t m e n t with i n d o m e t h a c i n also red u c e d in p a r t t h e m u c o s a l p r o t e c t i v e a n d b l o o d flow effects of sucralfate b u t t h e c o m b i n a t i o n o f ind o m e t h a c i n plus L - N N A c o m p l e t e l y a b o l i s h e d t h e s e effects it can b e c o n c l u d e d t h a t the N O a n d P G systems c o o p e r a t e in t h e m a i n t e n a n c e of a d e q u a t e m u c o s a l b l o o d flow a n d tissue integrity as s u g g e s t e d previously ( W h i t t l e et al., 1990).
3. Results References In a g r e e m e n t with p r e v i o u s findings, sucralfate conf e r r e d a l m o s t c o m p l e t e p r o t e c t i o n against the d a m a g e p r o d u c e d by e t h a n o l . This p r o t e c t i o n was r e v e r s e d in a d o s e - d e p e n d e n t m a n n e r by L - N N A , t h e d o s e e n h a n c ing the lesion a r e a to a b o u t 75% o f c o n t r o l v a l u e b e i n g a b o u t 50 m g / k g ( t a b l e 1). T h e inhibitory effect o f L - N N A was c o m p l e t e l y a n t a g o n i z e d by L - a r g i n i n e b u t not D - a r g i n i n e (100 m g / k g i.v.). T h e d a t a for t h e gross m u c o s a l d a m a g e w e r e p a r a l l e l e d by t h e c h a n g e s in gastric b l o o d flow. E t h a n o l a l o n e c a u s e d a b o u t 68%
Coleman, J.C., J.P Lacz, R.-K. Browne and D T Drees, 1987, Effects of sucralfate or mdd irritants on experimental gastritis and prostaglandm production, Am J Med. 83 (Suppl. 3B), 24 Hm, W.N., B.W. Chen, C.H. Cho, C.T Luk and S.K. Lam, 1991, Role of gastric mucosal blood flow m cytoprotection, Digestion 48, 113. Konturek, S.J., T Brzozowskl, D Drozdowlcz, E. Krzyzek, J Garhckl, J Majka, A. Dembmskl, J. Stachura and I. Amon, 1991, Nocloprost, a unique prostaglandm E analog with local gastroprotectwe and ulcer heahng activity, Eur. J Pharmacol. 195, 347
279 Konturek, S.J., T. Radeckl, I. Plastuckl, T Brzozowskl and D Drozdowlcz, 1987, GastroprotecUon by collo,dal bismuth subcltrate (De-Nol) and sucralfate. Role of endogenous prostaglandms, Gut 28, 210 Peskar, B M., W Respondek, K.M. Muller and B.A. Peskar, 1991, Role of mtnc oxide in capsalcm-mduced gastroprotectlon, Eur. J Pharmacol 198, 113
Whittle, B J R., J. Lopez-Belmonte and S. Moncada, 1990, Regulation of gastric mucosal integrity by endogenous mtrlc oxide interaction wRh prostanolds and sensory neuropeptides in the rat, Br J Pharmacol 99, 607.
277
© 1992 Elsevier Science Pubhshers B V. All rights reserved 0014-2999/92/$05.00
EJP 21000
Short communication
Role of nitric oxide and prostaglandins in sucralfate-induced gastroprotection Stanislaw J. K o n t u r e k , T o m a s z Brzozowski, J o l a n t a M a j k a a n d K r z y s z t o f Czarnobilski Instttute of Phystology, UnwersttyMedtcal School, Krakow, Poland Recewed 26 November 1991, accepted 10 December 1991
We investigated the role of nitric oxide (NO) and prostaglandins (PG) in the prevention by sucralfate of ethanol-induced gastric damage and the decrease of gastric blood flow and compared them with those obtained with nocloprost, a potent locally acting gastroprotective agent. Sucralfate and nocloprost given mtragastrically (i.g.) protected dose dependently the gastric mucosa against the damage by absolute ethanol and prevented the decrease in blood flow induced by ethanol. Pretreatment with NG-nltro-L-arglnine (L-NNA), an inhibitor of NO synthase decreased dose dependently the protection and the maintenance of blood flow provided by sucralfate but not by nocloprost. Th~s decrease of sucralfate protection was antagonized by L-arginine but not D-argmine. Pretreatment with indomethacin also reversed, in part, the protective and hyperemic effects of sucralfate but the combination of both indomethacln and L-NNA completely abolished these effects. We conclude that sucralfate activates both the NO and PG systems that cooperate in the gastroprotective action of this drug and that NO is not involved in the protection induced by a PGE: analog. Nitric oxide (NO); Gastroprotection; Blood flow (mucosal); Nocloprost
I. Introduction
2. Material and methods
Sucralfate, a complex of sucrose octasulfate and aluminium has been shown in numerous studies to prevent the formation of acute gastric mucosal lesions induced by various ulcerogens (Konturek et al., 1987), and this has been explained, in part, by the stimulation of prostaglandin (PG) biosynthesis by this drug. Recently, the increase in mucosal blood flow accompanying the gastroprotective action of capsaicin has been attributed to the biological activity of endothelium-derived relaxing factor, nitric oxide (NO), which was proposed to interact with mucosal prostanoids on both gastric blood flow and tissue integrity (Whittle et al., 1990; Peskar et al., 1991). We have, therefore, investigated the possible role of NO and PG in gastroprotection and gastric blood flow induced by sucralfate and compared their effect with that of nocloprost, a locally active gastroprotective and vasoactive agent (Konturek et al., 1991; Hui et al., 1991). For this purpose we used the inhibitor of NO biosynthesis, NG-nitro-L-arginine (L-NNA), and the substrate for NO synthase, Larginine (Sigma Chemicals Co., St. Louis, MO, U.S.A.).
Groups of 8-10 male Wistar rats (160-180 g) received L-NNA (12.5-50 m g / k g i.v.) a n d / o r indomethacin (5 m g / k g i.p.) or vehicle (saline, 1 ml/kg). Additional groups of rats received either L-arginine or D-arginine (100 m g / k g i.v. or the vehicle (1.0 m l / k g ) 15 min before L-NNA. About 15 min later sucralfate administered orally in a dose (100 m g / k g ) that we had found to induce almost complete gastroprotection against absolute ethanol (Konturek et al., 1987). In control experiments, 1 m l / k g of vehicle was used 15 min after pretreatment with L-NNA, or the combination of L-arginine or D-arginine plus L-NNA. In tests of the pretreatment with indomethacin, L-NNA (50 m g / k g i.v.) or sucralfate (100 m g / k g ) was administered and 100% ethanol was applied 30 min later. For comparison, a stable P G E 2 analog, nocloprost, was used instead of sucralfate at a dose (1 /zg/kg i.g. (intragastrically)) that was shown to induce almost complete gastroprotection against ethanol. A further 15 min after the application of sucralfate or nocloprost, 1.5 ml of 100% ethanol was instilled into the stomach. The rats were anesthetized 60 min later with urethane sodium pentobarbitone (60 m g / k g i.p); the abdomen was exposed by a midline incision and a small incision was made in the forestomach to allow emptying of
Correspondence to' S.J Konturek, Institute of Physiology, ul. Grzegorzecka 16, 31-531 Krakow, Poland
278 TABLE 1 Effects of L-NNA (50 mg/kg iv), indomethacm (5 mg/kg i.p), sucralfate (100 mg/kg Lg.) and nocloprost (1 /zg/kg i.g) with or without addition of L-argmme or D-arglmne (100 mg/kg) on the area of ethanol-reduced gastric lesion and gastric blood flow m rats Means+_S E.M. of 10-20 rats Lesion area
(mm2)
Blood flow (ml/min per 100 g)
Vehicle + ethanol (control)
88_+ 6
L-NNA+ ethanol
121 + 18 a
17+_3 a
17+_ 3 a
45+_7 a
68 +_ 7 b
22 +_4 b
20 +_ 4 a
37 +_5 a
76 5 : 8 a
20 +_3 b
94 +_19
17 +_3
38+ 4 b
31+5 b
Sucralfate+ethanol L-NNA + sucralfate + ethanol L-Arg + L-NNA + sucralfate + ethanol D-Arg + L-NNA + sucralfate + ethanol Indomethacm + vehicle + ethanol Indomethacln + sucralfate+ethanol Indomethacm + L-NNA +ethanol Indomethacm + L-NNA + sucralfate + ethanol Nocloprost +ethanol L-NNA + nocloprost + ethanol
23 +-2
127_+ 18 a
16+3
117_+27 b
19-+4 b
21 +_ 4 a
52+_7 a
28+_ 4 a
40+_5 a
a
a Slgmflcant (P < 0.05) change as compared to vehicle control values. b Sigmficant changes as compared with sucralfate + ethanol.
gastric c o n t e n t to t h e e x t e r i o r a n d for r e c o r d i n g o f gastric b l o o d flow using a laser D o p p l e r f l o w - m e t e r ( M o d e l B P M 403A, V a s a m e d i c s Inc., St. Paul, M N , USA). Finally, the s t o m a c h was r e m o v e d , o p e n e d a n d the a r e a o f gastric lesions was d e t e r m i n e d by p l a n i m e t r y . M e a n s ___S.E.M. w e r e c a l c u l a t e d for t h e lesion a r e a and m u c o s a l b l o o d flow. Statistical significance w e r e e v a l u a t e d using W i l c o x o n ' s r a n k test.
r e d u c t i o n in gastric b l o o d flow a n d t h e p r e t r e a t m e n t with L - N N A r e s u l t e d in a f u r t h e r significant d e c r e a s e o f this flow. L - A r g i n i n e (100 m g / k g ) b u t n o t D - a r g i n i n e given b e f o r e L - N N A (50 m g / k g ) p a r t l y r e s t o r e d the b l o o d flow r e d u c e d by t h e c o m b i n a t i o n o f L - N N A + e t h a n o l (table 1). P r e t r e a t m e n t with i n d o m e t h a c i n d i d n o t affect significantly t h e e t h a n o l - i n d u c e d gastric lesion a r e a or the b l o o d flow b u t r e v e r s e d in p a r t t h e p r o t e c t i v e effects o f sucralfate. T h e c o m b i n a t i o n o f i n d o m e t h a c i n plus LNNA completely abolished the mucosal protection and t h e rise in t h e b l o o d flow c a u s e d by sucralfate. A d m i n i s t r a t i o n o f n o c l o p r o s t (1 / x g / k g i.g.) gave a l m o s t c o m p l e t e p r e v e n t i o n of e t h a n o l - i n d u c e d d a m age a n d c h a n g e s in gastric b l o o d flow. P r e t r e a t m e n t with L - N N A (50 m g / k g ) o r the c o m b i n a t i o n of La r g i n i n e + L - N N A d i d not affect significantly t h e gast r o p r o t e c t i o n or m u c o s a l circulation a f t e r nocloprost.
4. Discussion T h e p r e s e n t results d e m o n s t r a t e indirectly, using a p o t e n t N O synthase i n h i b i t o r ( L - N N A ) , t h a t N O is involved in s u c r a l f a t e - i n d u c e d g a s t r o p r o t e c t i o n a n d suggest t h a t this involvement is r e s p o n s i b l e for the i n c r e a s e in m u c o s a l circulation. Since sucralfate is not a d s o r b e d from t h e g a s t r o i n t e s t i n a l l u m e n it r e m a i n s to be d e t e r m i n e d w h a t is t h e m e c h a n i s m o f activation o f t h e N O system by this drug. N o c l o p r o s t p r o t e c t e d the m u c o s a even a f t e r t h e b l o c k a d e o f N O synthase by L - N N A , i n d i c a t i n g t h a t N O is not involved in P G - i n d u c e d g a s t r o p r o t e c t i o n . Since t h e p r e t r e a t m e n t with i n d o m e t h a c i n also red u c e d in p a r t t h e m u c o s a l p r o t e c t i v e a n d b l o o d flow effects of sucralfate b u t t h e c o m b i n a t i o n o f ind o m e t h a c i n plus L - N N A c o m p l e t e l y a b o l i s h e d t h e s e effects it can b e c o n c l u d e d t h a t the N O a n d P G systems c o o p e r a t e in t h e m a i n t e n a n c e of a d e q u a t e m u c o s a l b l o o d flow a n d tissue integrity as s u g g e s t e d previously ( W h i t t l e et al., 1990).
3. Results References In a g r e e m e n t with p r e v i o u s findings, sucralfate conf e r r e d a l m o s t c o m p l e t e p r o t e c t i o n against the d a m a g e p r o d u c e d by e t h a n o l . This p r o t e c t i o n was r e v e r s e d in a d o s e - d e p e n d e n t m a n n e r by L - N N A , t h e d o s e e n h a n c ing the lesion a r e a to a b o u t 75% o f c o n t r o l v a l u e b e i n g a b o u t 50 m g / k g ( t a b l e 1). T h e inhibitory effect o f L - N N A was c o m p l e t e l y a n t a g o n i z e d by L - a r g i n i n e b u t not D - a r g i n i n e (100 m g / k g i.v.). T h e d a t a for t h e gross m u c o s a l d a m a g e w e r e p a r a l l e l e d by t h e c h a n g e s in gastric b l o o d flow. E t h a n o l a l o n e c a u s e d a b o u t 68%
Coleman, J.C., J.P Lacz, R.-K. Browne and D T Drees, 1987, Effects of sucralfate or mdd irritants on experimental gastritis and prostaglandm production, Am J Med. 83 (Suppl. 3B), 24 Hm, W.N., B.W. Chen, C.H. Cho, C.T Luk and S.K. Lam, 1991, Role of gastric mucosal blood flow m cytoprotection, Digestion 48, 113. Konturek, S.J., T Brzozowskl, D Drozdowlcz, E. Krzyzek, J Garhckl, J Majka, A. Dembmskl, J. Stachura and I. Amon, 1991, Nocloprost, a unique prostaglandm E analog with local gastroprotectwe and ulcer heahng activity, Eur. J Pharmacol. 195, 347
279 Konturek, S.J., T. Radeckl, I. Plastuckl, T Brzozowskl and D Drozdowlcz, 1987, GastroprotecUon by collo,dal bismuth subcltrate (De-Nol) and sucralfate. Role of endogenous prostaglandms, Gut 28, 210 Peskar, B M., W Respondek, K.M. Muller and B.A. Peskar, 1991, Role of mtnc oxide in capsalcm-mduced gastroprotectlon, Eur. J Pharmacol 198, 113
Whittle, B J R., J. Lopez-Belmonte and S. Moncada, 1990, Regulation of gastric mucosal integrity by endogenous mtrlc oxide interaction wRh prostanolds and sensory neuropeptides in the rat, Br J Pharmacol 99, 607.
