1. Introduction
Gastric mucosal integrity is maintained by multiple factors, including both paracrine and neuronal factors [1-4]. The former includes prostaglandins (PGs) [1] and nitric oxide (NO) [2], while capsaicin-sensitive afferent neurons play a central role in neuronal protection in the stomach [3]. Previous studies demonstrated that capsaicin, a selective stimulator of these afferent neurons, protected the gastric mucosa against various ulcerogenic stimuli such as nectrotizing agents [3]. The protective effects of capsaicin were shown to be mediated by these afferent neurons because they were completely attenuated by the chemical ablation of these neurons following a pretreatment with a large dose of capsaicin [3, 5]. The binding site of capsaicin has been cloned and named the transient receptor potential vaniloid type 1 receptor (TRPV1), a nonselective cationic channel [6]. Capsaicin is assumed to stimulate these afferent neurons by activating TRPV1, which results in the liberation of the neurotransmitter, calcitonin gene-related peptide (CGRP) and gastric protection.
Several studies, including our own, have showed that the protective effects of capsaicin were mitigated by the prior administration of indomethacin, which indicated the involvement of endogenous PGs in this action [6-9]. Endogenous PGs were previously shown to sensitize sensory neurons to nociceptive stimuli [10, 11]. Therefore, endogenous PGs are assumed to play a supportive role in the mechanism underlying capsaicin-induced gastric protection, possibly by sensitizing these afferent neurons, because capsaicin-induced gastric cytoprotection was shown to be attenuated by indomethacin.
On the other hand, recent pharmacological studies have classified PGE2 receptors into four specific G protein-coupled subtypes, EP1 to EP4 [12]. The distribution of these receptors is considered to explain the multiple effects of PGE2 in various tissues including the gastrointestinal tract. Mice lacking various receptors for prostanoids have been established [13, 14], and the roles of specific PG receptors in the various biological actions of PGs have been demonstrated using these "knockout mice" [15]. We performed a series of experiments to identify the EP receptor subtypes mediating the gastrointestinal protection afforded by PGE2 using various models in both rats and EP receptor knockout mice, and found that PGE2, administered exogenously or generated endogenously, provided gastric protection against HCl/ethanol mediated by EP1 receptors [16, 17]. However, the relationship between the EP receptor subtype and facilitation of capsaicin-induced gastric protection by PGs remains unknown.
We here investigated the role of endogenous PGs in the gastric protective action of capsaicin against HCl/ethanol-induced damage in rats, mainly in relation to PGE2 and EP receptors. Furthermore, because an animal model lacking various receptors for prostanoids is now available [13, 15, 18], we also evaluated the protective activity of capsaicin in knockout mice lacking EP1 or EP3 receptors and also in some cases IP receptors. In addition, we also examined the gastric hyperemic response to capsaicin in these knockout mice in order to provide functional evidence for the modulatory role of PGs in capsaicin-induced protective effects.
2. Methods
3. Results
3.1. Effect of capsaicin on HCl/ethanol-induced gastric lesions in rats
The oral administration of HCl/ethanol (60% in 150 mM HCl) produced multiple lesions in the glandular mucosa, along the long axis of the rat stomach. These lesions were dose-dependently prevented in animals pretreated with capsaicin (1-10 mg/kg) p.o. before the challenge with HCl/ethanol, and a significant effect was obtained at doses over 3 mg/kg, with the inhibition at 10 mg/kg being 81.6% (Figure 1A). The protective effects of capsaicin (10 mg/kg) were completely attenuated by the chemical ablation of sensory neurons as well as prior administration of indomethacin (5 mg/kg), but not by ONO-AE-829 (10 mg/kg)(Figure 1B). The severity of HCl/ethanol-induced gastric lesions was also significantly reduced by the prior i.v. administration of PGE2 (0.03 mg/kg), with the inhibition being 82.1% (Figure 2). The protective effects of PGE2 were significantly mitigated by the pretreatment with the EP1 antagonist, ONO-AE-829 (10 mg/kg), but not by chemical deafferentation. The degree of protection afforded by PGE2 in the presence of ONO-AE-829 at 10 mg/kg was 19.8%, which was significantly less than that observed in vehicle-treated normal rats.
The oral administration of capsaicin (10 mg/kg) did not affect the mucosal PGE2 content when measured 0.5 hr after its administration (not shown). The prior administration of indomethacin (5 mg/kg, s.c.) markedly reduced PGE2 levels in the presence of capsaicin. As in normal rats, capsaicin did not significantly affect mucosal PGE2 levels in sensory deafferented animals.
3.2. Reversal of capsaicin-induced protection by EP agonists in indomehtacin-pretreated rats
To investigate the roles of PGE2 and EP receptors in capsaicin-induced gastric protection, we examined the rescue effects of various subtype-specific EP agonists on capsaicin in the presence of indomethacin. The oral administration of capsaicin (10 mg/kg) markedly protected against HCl/ethanol-induced gastric lesions (Figure 3A). This effect of capsaicin was significantly mitigated by the prior administration of indomethacin (5 mg/kg), and the degree of inhibition was reduced to 29.7%. When these animals were given various EP agonists i.v. 20 min after indomethacin, the protective effects of capsaicin were again observed in rats pretreated with butaprost (an EP2 agonist). Neither ONO-NT-012 (an EP3 agonist) nor 11-deoxy PGE1 (an EP3/EP4 agonist) rescued the effects of capsaicin against HCl/ethanol in the presence of indomethacin. None of the EP agonists (i.v.) used, including butaprost, significantly protected against HCl/ethanol by themselves (Figure 3B).
3.3. Gastric cytoprotection against HCl/ethanol by capsaicin in mice
To further investigate the relationship between capsaicin-induced gastric protection and EP receptor subtype, we examined the protective effects of capsaicin against HCl/ethanol in both wild-type and knockout mice lacking EP1 or EP3 receptors. In addition, since other study reported a role for PGI2 in the release of CGRP in the stomach following capsaicin stimulation [19], the protective effect of capsaicin was also examined in IP receptor knockout mice. The intragastric administration of HCl/ethanol (0.3 ml) also caused hemorrhagic lesions in the mouse stomach (Figure 4). HCl/ethanol led to the development of gastric lesions in EP1, EP3, and IP receptor knockout mice, similar to wild-type mice, and the severity of these lesions was similar among these groups. The severity of these lesions in wild-type mice was also reduced by the prior p.o. administration of capsaicin (10 mg/kg). This agent significantly reduced the severity of these lesions in animals lacking either EP1 or EP3 receptors. However, capsaicin failed to protect the stomach against HCl/ethanol in IP receptor knockout mice, and the lesion score in these animals was not significantly different from that observed in IP receptor knockout animals without the capsaicin pretreatment.
The oral administration of capsaicin (10 mg/kg) did not significantly affect 6-keto PGF1α levels, which was similar to the effects observed on PGE2 content in the rat stomach (not shown). Indomethacin (5 mg/kg) markedly decreased 6-keto PGF1α levels in the presence of capsaicin. Similarly, capsaicin had no effect on 6-keto PGF1α levels in IP receptor knockout mice.
3.4. Effect of capsaicin on gastric mucosal blood flow in mice
Under urethane anesthesia, the chambered stomachs of both wild type mice and those lacking EP1, EP3, or IP receptors showed a relatively constant GMBF during a 2-hr test period. The mucosal application of capsaicin (1 mg/ml) for 10 min caused a marked increase in GMBF in wild type mice, and this effect was significantly attenuated by the prior administration of indomethacin (5 mg/ml) (Figure 5). A significant increase in GMBF by capsaicin was also observed in both EP1 and EP3 receptor knockout mice. However, the gastric hyperemic response to capsaicin was almost completely absent in animals lacking IP receptors, and GMBF values were significantly lower than those in control wild type mice, at most of the time points measured after the application of capsaicin.
4. Commentary
PGs, either endogenous or exogenous derivatives, have been shown to act on multiple receptors [12]. Capsaicin affords gastric protection by stimulating afferent C-fibers [3], and this action is partly dependent on endogenous PGs [5, 8, 9, 24]. We previously examined the relationship between EP receptor subtypes and gastric protection against HCl/ethanol in rats using various EP agonists and found that the gastroprotective action afforded by endogenous or exogenous PGs was mediated by EP1 receptors [16, 17]. However, the EP receptor subtypes or other prostanoid receptors responsible for this phenomenon has yet to be established. The present study was conducted to determine the prostanoid receptor(s) involved in capsaicin-induced gastric protection.
First, we confirmed that PGE2 prevented the development of HCl/ethanol-induced gastric lesions, and this action was attenuated by the EP1 antagonist, ONO-AE-829 [16]. This result was also verified in EP receptor knockout mice, with this protection being completely absent in mice lacking EP1 receptors [16, 17]. These results strongly suggest that the protective effects of exogenous PGE2 in the stomach were mainly mediated by the activation of EP1 receptors. On the other hand, endogenous PGs play a role in the gastric cytoprotection induced by the oral administration of capsaicin [5, 8, 9]. As shown in this study, the protective effects of capsaicin against HCl/ethanol were dose-dependent, and were attenuated by the chemical ablation of capsaicin-sensitive sensory neurons. The protective effects of capsaicin were also significantly mitigated by the prior administration of indomethacin, which indicated the involvement of endogenous PGs in these effects. However, in contrast to the adaptive cytoprotection induced by a mild irritant [17], the effects of capsaicin were not affected by the selective EP1 antagonist, ONO-AE-829. This was confirmed by the stimulatory effects of capsaicin on gastric HCO3-secretion which were attenuated by indomethacin and capsazepine, but not ONO-8711 (an EP1 antagonist) [25]. Furthermore, neither the stimulation of sensory neurons by capsaicin nor sensory deafferentation affected mucosal PGE2 levels in the stomach. Many studies have shown that mild irritants increased the production of PGE2 in the stomach [14, 17]. These findings suggest that although endogenous PGs are involved in the gastric cytoprotection induced by both mild irritants and capsaicin, the mode of action appears to be different in these two cases. The stimulation of afferent neurons by capsaicin was assumed to increase the production of PGs in the stomach, but exerted protective effects in the stomach, that were partly dependent on endogenous PGs.
In the present study, we administered various EP agonists to indomethacin-treated animals, to determine whether the inhibitory effect of indomethacin on capsaicin-induced gastric protection was reversed by exogenous PGE2, and if so, which EP receptor subtype was responsible for this action. The protective effects of capsaicin were significantly restored, even in the presence of indomethacin, by the prior administration of butaprost, the EP2 agonist, but not by the EP3 or EP4 agonist. In addition, the protective effects of capsaicin were significantly enhanced in the presence of butaprost, which strongly suggested a supportive role for EP2 receptors in capsaicin-induced gastric protection. These results are supported by the findings of Haupt et al [26], who reported the involvement of the EP2 receptor in the potentiation of afferent neuronal discharges by PGE2 in the rat jejunum. Jenkins et al [27] also demonstrated that the activation of DP, EP, and IP receptors could each cause the release of CGRP from trigeminal neurons, and that the predominant EP receptor subtype involved may be the EP2 receptor. In the present study, neither of these EP agonists, including butaprost, offered any protection against HCl/ethanol-induced gastric damage by themselves. Furthermore, capsaicin-induced gastric protection was not affected by the EP1 antagonist, which excluded the involvement of EP1 receptors in the facilitation of this action by endogenous PGs. The significant level of protection afforded by capsaicin was also observed in knockout mice lacking EP1 and EP3 receptors, which confirmed that capsaicin-induced gastric protection did not involve EP1 or EP3 receptors. We could not confirm the involvement of EP2 receptors in this action because EP2 knockout mice were not available in our laboratory.
In contrast, we demonstrated that capsaicin failed to exhibit a cytoprotective effect against HCl/ethanol-induced gastric lesions in IP-receptor knockout mice. We previously showed that 20 mM taurocholate, as a mild irritant, protected the stomach against HCl/ethanol even in IP receptor knockout mice, which was similar to that observed in wild-type mice. These results suggested the absence of the involvement of PGI2 in the mechanism responsible for adapative cytoprotection [17]. We also reported that the adaptive cytoprotection induced by taurocholate was attenuated by ONO-AE-829, the EP1 antagonist, as well as indomethacin, and was not observed in EP1 receptor knockout mice [17]. The present results obtained in knockout mice suggest that IP receptors are also involved in the protective effects of capsaicin in the stomach, in addition to EP2 receptors. The exact mechanism by which endogenous PGs contributes to the protective action of capsaicin is currently unknown. Previous studies have suggested that endogenous PGs may sensitize sensory neurons to nociceptive stimuli [10, 11]. Boku et al. [19] reported the lack of CGRP release in response to mild injuries in the stomachs of IP-receptor knockout mice. Oishi et al. [10] demonstrated, using IP-receptor knockout mice, that PGI2 was a major nociceptive mediator in the acetic acid-induced writhing reaction. Since capsaicin-induced gastric cytoprotection was attenuated by indomethacin and was absent in IP-receptor knockout mice, endogenous PGI2 may play a supportive role in the mechanism responsible for capsaicin-induced gastric cytoprotection, possibly by sensitizing sensory neurons [17, 28]. However, capsaicin did not have any effect on either PGE2 production in the rat stomach or PGI2 production in the mouse stomach. Thus, PGs generated constitutively may maintain the sensitivity of these neurons to the capsaicin stimulation.
Intragastric capsaicin has been shown to increase GMBF in the rat stomach, and this effect was attenuated by sensory deafferentation following a capsaicin pretreatment [9, 14, 29]. Although gastric hyperemia is not the exclusive mechanism responsible for gastric cytoprotection induced by PGE2 or the stimulation of capsaicin-sensitive afferent neurons [16, 30], GMBF is considered to be a factor in capsaicin-induced gastric protection under certain experimental conditions [7, 29]. We previously reported that the gastric hyperemic response to capsaicin was also significantly mitigated by indomethacin, which suggested the involvement of endogenous PGs in this response [14]. In the present study, we confirmed that intragastric capsaicin markedly increased GMBF in wild type mice in an indomethacin-sensitive manner. This effect was also observed in EP1 or EP3 receptor knockout mice, but was completely absent in animals lacking IP receptors, similar to the gastroprotective effects of capsaicin. These results may provide functional evidence for the modulatory role of IP receptors in the facilitation of gastric protection mediated by capsaicin-sensitive afferent neurons by endogenous PGs.
The results of the present study suggest that capsaicin exhibits gastric cytoprotective effects as well as gastric hyperemic response, essentially by stimulating sensory neurons, and this partly depended on endogenous PG levels. The facilitative effects of endogenous PGs were mediated by EP2 and IP receptors, which may have sensitized the sensory neurons to capsaicin, even though capsaicin increased the production of PGI2 but not PGE2 in the gastric mucosa. However, whether endogenous PGs modulated the effects of capsaicin by interacting with TRPV1 remains unknown; however, capsaicin previously exhibited gastric protective effects by activating TRPV1 [31]. Endogenous phosphatidyl-inositol-4, 5-bisphosphate (PtdIns(4, 5) P2) was shown to inhibit TRPV1, and this was alleviated by agents that activated phospholipase C [32, 33]. Therefore, PGs may sensitize these afferent neurons to capsaicin through EP2/IP receptors by somehow releasing TRPV1 from PtdIns(4, 5)P2-mediated inhibition.
5. Summary
Capsaicin provided gastric cytoprotection essentially through the stimulation of sensory neurons, and this partly depended on endogenous PGs. PGs facilitated the protective effects of capsaicin, and this response was mediated by EP2 and IP receptors, possibly by sensitizing the sensory neurons to capsaicin, even though capsaicin increased the production of PGI2 but not PGE2 in the gastric mucosa (Figure 6). Although endogenous PGs are also known to be involved in the adaptive cytoprotection induced by mild irritants, this is different from that of capsaicin and is mediated by the activation of EP1 receptors with a concomitant increase in the production of mucosal PGE2.
Acknowledgments
The authors are greatly indebted to Professor Shu Narumiya, Kyoto University Faculty of Medicine, for kindly supplying EP1, EP3, and IP receptor-knockout mice and Ono Pharmaceutical Company for supplying various EP agonists and antagonists. We also thank the students at the Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan, for their technical collaboration.
References
- 1.
Robert A, Nezamis JE, Lancaster C, Davis JP, Field SO, Hanchar AJ. Mild irritants prevent gastric necrosis through "adaptive cytoprotection" mediated by prostaglandins. Am J Physiol 1983; 245: G113-21. - 2.
Whittle BJR, Lopez-Belmote J, Moncada S. Regulation of gastric mucosal integrity by endogenous nitric oxide: Interaction with prostaglandins and sensory neuropeptides in the rat. Br J Pharmacol 1990; 99: 607-11. - 3.
Holzer P, Sametz W. Gastric mucosal protection against ulcerogenic factors in the rat mediated by capsaicin-sensitive afferent neurons. Gastroenterology 1986; 91: 975-81. - 4.
Holzer P. Neural emergency system in the stomach. Gastroenterology 1998; 114: 823-39. - 5.
Takeuchi K, Niida H, Matsumoto J, Ueshima K, Okabe S. Gastric motility changes in capsaicin-induced cytoprotection in the rat stomach. Jpn J Pharmacol 1991; 55: 147-55. - 6.
Caterina MJ, Schumacher MA, Tominaga M, Rosen T, Levine JD, Lulius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997; 389: 316-24. - 7.
Takeuchi K, Ohuchi T, Narita M, Okabe S. Capsaicin-sensitve sensory nerves in recovery of gastric mucosal integrity after damage by sodium taurocholate in rats. Jpn J Pharmacol 1993; 63: 479-85. - 8.
Uchida M, Yano S, Watanabe K. The role of capsaicin-sensitive afferent nerves in protective effect of capsaicin against absolute ethanol-induced gastric lesions in rats. Jpn J Pharmacol 1991; 55: 279-82. - 9.
Brzozowski T, Drozdowicz D, Szlachcic A, Pytko-Polonczyk J, Majka J, Konturek SJ. Role of nitric oxide and prostaglandins in gastroprotection induced by capsaicin and papaverine. Digestion 1993; 54: 24-31. - 10.
Ohishi S, Ueno A, Matsumoto H, Murata T, Ushikubi F, Narumiya S. Evidence for involvement of prostaglandin I2 as a major nociceptive mediator in acetic acid-induced writhing reaction: a study using IP-receptor disrupted mice. Adv Exp Med Biol 1999; 469: 265-8. - 11.
Ueno A, Naraba H, Ikeda Y, Ushikubi F, Murata T, Narumiya S, Ohishi S. Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: a study on the paw edema induced in IP-receptor-deficient mice. Life Sci 2000; 66: PL155-60. - 12.
Coleman RA, Smith WL, Narumiya S. Classification of prostanoid receptors: Properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994; 46: 205-29. - 13.
Sugimoto Y, Namba T, Honda A, Hayashi Y, Negishi M, Ichikawa A, Narumiya S. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol Chem 1992; 267: 6463-6. - 14.
Matsumoto J, Takeuchi K, Ueshima K, Okabe S. Role of capsaicin-sensitive afferent neurons in mucosal blood flow response of rat stomach induced by mild irritants. Dig Dis Sci 1992; 37: 1336-44. - 15.
Ushikubi F, Segi E, Sugimoto Y, Murata T, Matsuoka T, Kobayashi T, Hizaki H, Tuboi K, Katsuyama M, Ichikawa A, Tanaka T, Yoshida N and Narumiya S. Impaired febrile response in mice lacking the prostaglandin E receptor subtype 3. Nature 1998; 395: 281-4 - 16.
Araki H, Yagi K, Suzuki K, Furukawa O, Takeuchi K. Roles of prostaglandin E receptor subtypes in cytoprotective action of prostaglandin E2 in rat stomachs. Alimental Pharmacol Ther 2000; 14 (Suppl 1): 18-25. - 17.
Takeuchi K, Araki H, Umeda M, Komoike Y, Suzuki K. Adaptive gastric cytoprotection is mediated by prostaglandin EP1 receptors: A study using rats and knockout mice. J Pharmacol Exp Ther 2001; 297: 1160-5. - 18.
Oida H, Namba T, Sugimoto Y, Ushikubi F, Ohishi H, Ichikawa A, Narumiya S. In situhybridization studies of prostacyclin receptor mRNA expression in various mouse organs. Br J Pharmacol 1995; 116: 2828-37. - 19.
Boku K, Ohno T, Saeki T, Hayashi H, Hayashi I, Katori M, Murata T, Narumiya S, Saigenji K, Majima M. Adaptive cytoprotection mediated by prostaglandin I2 is attributable to sensitization of CRGP-containing sensory nerves. Gastroenterology 2001; 120: 134-43. - 20.
Watabe A, Sugimoto Y, Honda A, Irie A, Namba T, Negishi M, Ito S, Narumiya S, Ichikawa A. Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor. J Biol Chem 1993; 27: 20175-8. - 21.
Takeuchi K, Komoike Y, Takeeda M, Ukawa H. Gastric mucosal ulcerogenic responses following barrier disruption in knockout mice lacking prostaglandin EP1 receptors. Aliment Pharmacol Ther 2002; 16: 74-82. - 22.
Futaki N, Takahashi S, Yokoyama M, Arai I, Higuchi S, Otomo S. NS-398, a new antiinflammatory agent, selectively inhibits prostaglandin G/H synthase/ cyclooxygenase (COX-2) activity in vitro. Prostaglandins 1994; 47: 55-9 - 23.
Holzer P, Livingston EH, Saria A, Guth PH. Sensory neurons mediate protective vasodilatation in rat gastric mucosa. Am J Physiol 1991; 260: G363-70. - 24.
Takeuchi K, Matsumoto J, Ueshima K, Okabe S. Role of capsaicin-sensitive afferent neurons in alkaline secretory response to luminal acid in the rat duodenum. Gastroenterology 1991; 101: 954-61. - 25.
Aihara E, Hayashi M, Sasaki Y, Kobata A, Takeuchi K. Mechanisms underlying capsaicin-stimulated HCO3-secretion in the stomach: Comparison with mucosal acidification. J Pharmacol Exp Ther 2005; 315: 423-32. - 26.
Haupt W, Jiang W, Kreis ME, Grundy D. Prostaglandin EP receptor subtypes have distinctive effects on jejunal afferent sensitivity in the rat. Gastroenterology 2000; 119: 1580-9. - 27.
Jenkins DW, Feniuk W, Humphrey PPA. Characterization of the prostanoid receptor types involved in mediating calcitonin gene-related peptide release from cultured rat trigeminal neurons. Br J Pharmacol 2001; 134: 1296-302. - 28.
Fukushima K, Aoi Y, Kato S, Takeuchi K. Gastroprotective action of lafutidine mediated by capsaicin-sensitive afferent neurons without interaction with TRPV1 and involvement of endogenous prostaglandins. World J Gastroenterol 2006; 12: 3031-37. - 29.
Takeuchi K, Kato S, Ogawa Y, Kanatsu K, Umeda M. Role of endogenous prostacyclin in gastric ulcerogenic and healing responses: A study using IP-receptor knockout mice. J Physiol Paris 2001; 95: 75-80. - 30.
Stroff T, Plate S, Ebrahim JS, Ehrlich KH, Respondek M, Peskar BM. Tachykinin-induced increase in gastric mucosal resistance: role of primary afferent neurons, CGRP, and NO. Am J Physiol 1996; 271: G1017-27. - 31.
Yamamoto H, Horie S, Uchida M, Tsuchiya S, Murayama T, Watanabe K. Effects of vanilloid receptor agonists and antagonists on gastric antral ulcers in rats. Eur J Pharmacol 2001; 432: 203-10. - 32.
Premkumar LS, Ahern GP. Induction of vanilloid receptor channel activity by protein kinase C. Nature 2000; 408: 985-90. - 33.
Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, Julius D. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4, 5)P2-mediated inhibition. Nature 2001; 411: 957-62.