European Journal of Pharmacology, 198 (1991) 113-114
113
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100408J
EJP 0304R
Rapid communication
A role for nitric oxide in capsaicin-induced gastroprotection Brigitta M. Peskar 1 Michael R e s p o n d e k 2 Klaus M. Miiller 2 and Bernhard A. Peskar 3 / Department of Experimental Clinical Medicine, 2 Department of Pathology, University Hospital Bergmannsheil and 3 Department of Pharmacology and Toxicology, Ruhr-University of Bochum, D-4630 Bochum, F.R.G. Received 29 April 1991, accepted 1 May 1991
Stimulation of afferent sensory nerve endings by intragastric administration of capsaicin protects against ethanol-induced rat gastric mucosal lesions (for review see Holzer, 1988). The primary mechanism of afferent nerve-mediated protection seems to be an increase in local blood flow (Holzer et al., 1990). This effect is not mediated by prostaglandins (PG) as the release of eicosanoids from gastric mucosa! fragments ex vivo is not increased by capsaicin treatment and the cyclooxygenase inhibitor, indomethacin, has no effect on capsaicin-induced protection (Holzer et al., 1990). We have now investigated the possible role of the potent endogenous vasodilator, nitric oxide (NO), in capsaicin-induced gastric mucosal protection. For this purpose we have used the inhibitor of NO biosynthesis, NG-nitro-L-arginine (L-NNA, Sigma Chemicals Co., St. Louis, MO, USA). Groups of 5-7 male Wistar rats (180-220 g) received intravenous injections of L-NNA (0.5-5 mg/kg) or the solvent, saline (2.0 ml/kg). Additional groups of rats received immediately before L-NNA either Larginine or D-arginine (100 mg/kg i.v. each) or the solvent saline (2.0 ml/kg). Ten minutes later 0.5 mg/kg capsaicin, suspended in 0.25% carboxymethylcellulose, was administered orally. The controls received 2 ml/kg of vehicle. After a further 30 min 1.5 ml of absolute ethanol was instilled into the stomach. The rats were killed 5 min later, the stomachs were removed, opened and a lesion index of gross mucosal damage was determined as described previously (Holzer et al., 1990). Some of the stomachs were fixed in 5% buffered formalin. A full length portion of tissue cut from the glandular mucosa was stained with hematoxylin/eosin and examined under a light microscope. The severity of mucosal injury was evaluated using a scoring system ranging from 1 (epithelial cell damage) to 4 (deep
Correspondence to: B.A. Peskar, Department of Pharmacology and Toxicology, Ruhr-University of Bochum, Universit[itsstrasse 150, D4630 Bochum, F.R.G.
necrosis and ulceration). The histological index (HI) was calculated as the percentage of total section-length of each grade of damage multiplied by the respective severity factor, the maximum (100% grade 4) thus being 400. Means + S.E.M. were calculated. Statistical significances were evaluated using the Wilcoxon rank test. In agreement with data of Holzer (1988) it was found that capsaicin protects the rat gastric mucosa against ethanol-induced damage (fig. 1). L-NNA increased mucosal damage significantly and abolished theprotective effect of capsaicin (fig. 1). The effect of L-NNA on capsaicin-induced gastroprotection was dose-dependent. Doses as low as 0.5 mg/kg and 1.5 mg/kg reduced the protective effect of capsaicin by 24 + 9 and 81 + 4%, respectively. L-Arginine, but not D-arginine, antagonized the effect of L-NNA (fig. 1).
75.Lesion index
50"
"A"
%'~'N xx~l ....
Co L-NNA Caps
@
+ L-Arq D-Aro L-NNA + Caps
Fig. 1. Effects of NG-nitrooL-arginine (L-NNA, 5 m g / k g i.v.), intragastric capsaicin (Caps, 0.5 mg/kg) and of combined treatment with these compounds on ethanol-induced rat gastric mucosal damage as compared to controls (Co). L-Arginine (L-Arg, 100 m g / k g i.v.), but not D-arginine (D-Arg, 100 m g / k g i.v.) antagonized the inhibition of capsaicin-induced protection by L-NNA. * P < 0.02, A p < 0.001 vs. controls, • p < 0.001 vs. capsaicin alone, ! p < 0.001 vs. capsaicin plus L-NNA; n = 5-7 per group.
The data on gross mucosal damage were paralleled by the results of histological examination. Capsaicin reduced the Hi from 221 + 9 in controls to 143 + 12 ( P < 0.001). Pretreatment with L-NNA (5 m g / k g ) reversed the HI to 208 + 3 ( P < 0.001 vs. capsaicin alone). Administration of L-arginine (100 m g / k g ) simultaneously with 5 m g / k g L-NNA completely restored the protection by capsaicin (HI 140 + 17), while D-arginine was inactive (HI 206 + 20). The results showed indirectly, by use of an inhibitor of biosvnthesis, that NO is involved in capsaicin-induced gastroproteetion. The capsaicin effect seems to be mediated by local release of sensory neuropeptides (Holzer, 1988). Neuropeptides could in turn release NO. The existence of such a mechanism is supported by the finding that substance P induces endotheliumdependent vasodilation (Furchgott, 1983). It remains, however, to be established that protective neuropeptides, in fact, release NO. Alternatively, capsaicin might release NO from gastric mucosal nerve endings directly. In this context it is of interest that NO has been shown to be a neurotransmitter in non-adrenergic non-cholinergic nerves (Bult et al., 1990). The effect of L-NNA on capsaicin-mediated gastroprotection is relatively selective. Thus, the gastroprotection provided by the anti-ulcer drug, carbenoxolone (200 m g / k g orally), is fully antagonized only by a dose of L-NNA as high as 40 mg/kg, while L-NNA 5 m g / k g has no effect (unpublished results). It has been demonstrated recently
(Whittle et al., 1990) that afferent sensory neurons and NO interact to preserve gastric mucosal integrity under basal conditions. Our results show that endogenous NO has, in addition, a mediator role in capsaicin-induced gastroprotection.
Acknowledgement We thank Mr. K.H. Ehrlich for excellent technical assistance.
References Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van ~!aercke and A.G. Herman, 1990, Nitric oxide as an inhibitc, y non-adrenergic non-cholinergic neurotransmitter, Nature 345, 346. Furchgott, R,F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Hoizer, P., 1988, Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin generelated peptide and other neuropeptides, Neuroscience 24, 739. Holzer, P., M.A. Pabst, l.Th. Lippe, B.M. Peskar, B.A. Peskar, E.H. Livingston and P.H. Guth, 1990, Afferent nerve-mediated protection against deep mucosal damage in the rat stomach, Gastroenterology 98, 838. Whittle, B.J.R., J. Lopez-Belmonte and S. Moncada, 1990, Regulation of gastric mucosal integrity by endogenous nitric oxide: interactions with prostanoids and sensory neuropeptides in the rat, Br. J. Pharmacol. 99, 607.
113
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100408J
EJP 0304R
Rapid communication
A role for nitric oxide in capsaicin-induced gastroprotection Brigitta M. Peskar 1 Michael R e s p o n d e k 2 Klaus M. Miiller 2 and Bernhard A. Peskar 3 / Department of Experimental Clinical Medicine, 2 Department of Pathology, University Hospital Bergmannsheil and 3 Department of Pharmacology and Toxicology, Ruhr-University of Bochum, D-4630 Bochum, F.R.G. Received 29 April 1991, accepted 1 May 1991
Stimulation of afferent sensory nerve endings by intragastric administration of capsaicin protects against ethanol-induced rat gastric mucosal lesions (for review see Holzer, 1988). The primary mechanism of afferent nerve-mediated protection seems to be an increase in local blood flow (Holzer et al., 1990). This effect is not mediated by prostaglandins (PG) as the release of eicosanoids from gastric mucosa! fragments ex vivo is not increased by capsaicin treatment and the cyclooxygenase inhibitor, indomethacin, has no effect on capsaicin-induced protection (Holzer et al., 1990). We have now investigated the possible role of the potent endogenous vasodilator, nitric oxide (NO), in capsaicin-induced gastric mucosal protection. For this purpose we have used the inhibitor of NO biosynthesis, NG-nitro-L-arginine (L-NNA, Sigma Chemicals Co., St. Louis, MO, USA). Groups of 5-7 male Wistar rats (180-220 g) received intravenous injections of L-NNA (0.5-5 mg/kg) or the solvent, saline (2.0 ml/kg). Additional groups of rats received immediately before L-NNA either Larginine or D-arginine (100 mg/kg i.v. each) or the solvent saline (2.0 ml/kg). Ten minutes later 0.5 mg/kg capsaicin, suspended in 0.25% carboxymethylcellulose, was administered orally. The controls received 2 ml/kg of vehicle. After a further 30 min 1.5 ml of absolute ethanol was instilled into the stomach. The rats were killed 5 min later, the stomachs were removed, opened and a lesion index of gross mucosal damage was determined as described previously (Holzer et al., 1990). Some of the stomachs were fixed in 5% buffered formalin. A full length portion of tissue cut from the glandular mucosa was stained with hematoxylin/eosin and examined under a light microscope. The severity of mucosal injury was evaluated using a scoring system ranging from 1 (epithelial cell damage) to 4 (deep
Correspondence to: B.A. Peskar, Department of Pharmacology and Toxicology, Ruhr-University of Bochum, Universit[itsstrasse 150, D4630 Bochum, F.R.G.
necrosis and ulceration). The histological index (HI) was calculated as the percentage of total section-length of each grade of damage multiplied by the respective severity factor, the maximum (100% grade 4) thus being 400. Means + S.E.M. were calculated. Statistical significances were evaluated using the Wilcoxon rank test. In agreement with data of Holzer (1988) it was found that capsaicin protects the rat gastric mucosa against ethanol-induced damage (fig. 1). L-NNA increased mucosal damage significantly and abolished theprotective effect of capsaicin (fig. 1). The effect of L-NNA on capsaicin-induced gastroprotection was dose-dependent. Doses as low as 0.5 mg/kg and 1.5 mg/kg reduced the protective effect of capsaicin by 24 + 9 and 81 + 4%, respectively. L-Arginine, but not D-arginine, antagonized the effect of L-NNA (fig. 1).
75.Lesion index
50"
"A"
%'~'N xx~l ....
Co L-NNA Caps
@
+ L-Arq D-Aro L-NNA + Caps
Fig. 1. Effects of NG-nitrooL-arginine (L-NNA, 5 m g / k g i.v.), intragastric capsaicin (Caps, 0.5 mg/kg) and of combined treatment with these compounds on ethanol-induced rat gastric mucosal damage as compared to controls (Co). L-Arginine (L-Arg, 100 m g / k g i.v.), but not D-arginine (D-Arg, 100 m g / k g i.v.) antagonized the inhibition of capsaicin-induced protection by L-NNA. * P < 0.02, A p < 0.001 vs. controls, • p < 0.001 vs. capsaicin alone, ! p < 0.001 vs. capsaicin plus L-NNA; n = 5-7 per group.
The data on gross mucosal damage were paralleled by the results of histological examination. Capsaicin reduced the Hi from 221 + 9 in controls to 143 + 12 ( P < 0.001). Pretreatment with L-NNA (5 m g / k g ) reversed the HI to 208 + 3 ( P < 0.001 vs. capsaicin alone). Administration of L-arginine (100 m g / k g ) simultaneously with 5 m g / k g L-NNA completely restored the protection by capsaicin (HI 140 + 17), while D-arginine was inactive (HI 206 + 20). The results showed indirectly, by use of an inhibitor of biosvnthesis, that NO is involved in capsaicin-induced gastroproteetion. The capsaicin effect seems to be mediated by local release of sensory neuropeptides (Holzer, 1988). Neuropeptides could in turn release NO. The existence of such a mechanism is supported by the finding that substance P induces endotheliumdependent vasodilation (Furchgott, 1983). It remains, however, to be established that protective neuropeptides, in fact, release NO. Alternatively, capsaicin might release NO from gastric mucosal nerve endings directly. In this context it is of interest that NO has been shown to be a neurotransmitter in non-adrenergic non-cholinergic nerves (Bult et al., 1990). The effect of L-NNA on capsaicin-mediated gastroprotection is relatively selective. Thus, the gastroprotection provided by the anti-ulcer drug, carbenoxolone (200 m g / k g orally), is fully antagonized only by a dose of L-NNA as high as 40 mg/kg, while L-NNA 5 m g / k g has no effect (unpublished results). It has been demonstrated recently
(Whittle et al., 1990) that afferent sensory neurons and NO interact to preserve gastric mucosal integrity under basal conditions. Our results show that endogenous NO has, in addition, a mediator role in capsaicin-induced gastroprotection.
Acknowledgement We thank Mr. K.H. Ehrlich for excellent technical assistance.
References Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van ~!aercke and A.G. Herman, 1990, Nitric oxide as an inhibitc, y non-adrenergic non-cholinergic neurotransmitter, Nature 345, 346. Furchgott, R,F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Hoizer, P., 1988, Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin generelated peptide and other neuropeptides, Neuroscience 24, 739. Holzer, P., M.A. Pabst, l.Th. Lippe, B.M. Peskar, B.A. Peskar, E.H. Livingston and P.H. Guth, 1990, Afferent nerve-mediated protection against deep mucosal damage in the rat stomach, Gastroenterology 98, 838. Whittle, B.J.R., J. Lopez-Belmonte and S. Moncada, 1990, Regulation of gastric mucosal integrity by endogenous nitric oxide: interactions with prostanoids and sensory neuropeptides in the rat, Br. J. Pharmacol. 99, 607.
