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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan
Received May 26, 2003; accepted August 8, 2003.
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Abstract |
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). The key enzyme in the pathway for PG synthesis, cyclooxygenase (COX), exists as two isozymes referred to as COX-1 and COX-2. COX-1, constitutively expressed in normal gastric mucosa, has been proposed to generate PGs involved in the maintenance of essential physiological functions (Simmons et al., 1991; O'Neill and Ford-Hutchinson, 1993), whereas COX-2, characterized by a rapid inducibility in response to various proinflammatory stimuli, has been thought to be responsible for pathological PG production at inflammatory sites (O'Banion et al., 1991; Xie et al., 1992). Studies using selective COX-2 inhibitors showed that the gastric ulcerogenic property of nonsteroidal antiinflammatory drugs (NSAIDs) is brought about by inhibition of COX-1 but not COX-2 (Whittle, 1983; Futaki et al., 1993). However, it has been recently shown that inhibition of both COX-1 and COX-2 is required for NSAID-induced gastric injury, suggesting a role for COX-2 as well as COX-1 in maintaining the integrity of the stomach (Wallace et al., 2000; Tanaka et al., 2001).
It is known that the application of mild irritants to the stomach causes an increase in gastric mucosal blood flow (GMBF) as well as a decrease in acid secretion (Whittle, 1983; Takeuchi et al., 1986, 1993). These agents damage the surface epithelium of the gastric mucosa, resulting in acid back-diffusion, yet they rarely cause macroscopically visible damage and actually protect the stomach against necrotizing agents (Holzer et al., 1991; Takeuchi et al., 1993). Such hyperemic responses subside in the presence of NSAIDs, suggesting a role for endogenous PGs in this phenomenon (Whittle, 1983; Nobuhara and Takeuchi, 1984; Takeuchi et al., 1986, 1987). We previously reported, using a selective COX-2 inhibitor, that endogenous PGs produced by COX-1 play an important role in maintaining the gastric hyperemic response and mucosal integrity after barrier disruption by taurocholate (TC) Na (Hirata et al., 1997), yet the relative contribution of the COX-1 and COX-2 isozymes to the maintenance of other functional responses such as acid secretion in the stomach after barrier disruption is not entirely clear.
In the present study, we examined the effects of selective COX-1 and COX-2 inhibitors on changes in various functions of the rat stomach after exposure to TC and investigated the role of COX isozymes in functional responses of the stomach under adverse conditions, i.e., after barrier disruption.
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Materials and Methods |
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200 to 230 g (Charles River, Shizuoka, Japan), were used in all experiments. The animals were kept in individual cages with raised mesh bottoms and deprived of food but allowed free access to tap water for 18 h before the experiments. Studies were carried out using four to six rats per group. All experimental procedures described here were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University.
Experimental Protocol
The experiments were performed in four groups of rats; each group was pretreated with saline, indomethacin (a nonselective COX-1 and COX-2 inhibitor), SC-560 (a selective COX-1 inhibitor) (Smith et al., 1998), and rofecoxib (a selective COX-2 inhibitor) (Chan et al., 1995), respectively. In these groups of rats, the effects of mucosal application of 20 mM TC plus 50 mM HCl on gastric transmucosal potential difference (PD), GMBF, luminal acid loss (acid back-diffusion), acid secretion, and PGE2 production were examined under urethane anesthesia. Various COX inhibitors were given intraduodenally at a dose of 5 mg/kg, 30 min before the TC treatment. In some rats, the expression of mRNA for COX-1 and COX-2 was examined using reverse transcription-polymerase chain reaction in the stomach after exposure to TC. In a separate study, we also examined the effects of these COX inhibitors on the production of PGE2 in carrageenan-induced paw edema in rats.
Determination of PD, GMBF, and Acid Back-Diffusion
Animals were anesthetized with urethane (1.25 g/kg, intraperitoneally), and the trachea was cannulated to ensure a patent airway. Acid secretion was completely inhibited by pretreatment with omeprazole (60 mg/kg, intraperitoneally). Simultaneous measurement of PD, GMBF, and acid back-diffusion was performed in a chambered stomach as described previously (Matsumoto et al., 1992; Takeuchi et al., 1994). Briefly, the abdomen was incised, and the stomach was exposed and mounted on an ex vivo chamber (area exposed, 3.14 cm2). At the beginning of each experiment, the mucosa was rinsed several times with a solution of 50 mM HCl plus 100 mM NaCl. When the gastric exudate became clear, 2 ml of the acid solution was instilled in the chamber, and 15 min later the gastric contents were recovered from the chamber. This procedure was repeated every 15 min, two times before and six times after exposure of the mucosa to 20 mM TC plus 50 mM HCl for 30 min. PD was determined using two agar bridges, one positioned in the chamber and the other in the abdominal cavity. GMBF was measured with a laser Doppler flow-meter (model ALF 21; Advance, Tokyo, Japan), placing the probe gently on the corpus mucosa using a balancer, and changes in GMBF were continuously monitored on a two-channel recorder (U-228; Tokai-Irika, Tokyo, Japan) simultaneously with those of PD. Acid back-diffusion (luminal acid loss) was determined from analyses of the collected acid solution. Each sample was analyzed for volume and acid concentration, which was determined by automatic titration of an aliquot with 0.1 N NaOH to pH 7.0 (Autoburette, Comtite-7; Hiranuma, Tokyo, Japan). The amount of acid back-diffusion was calculated as the difference between the product of the final volume and concentration and the product of the initial volume and concentration. Positive values indicate that the net flux of H+ was from the mucosa to the lumen, and the results were expressed as microequivalents per 15 min. At the end of each experiment, i.e., 90 min after exposure to TC, the mucosa was examined for hemorrhagic damage under a dissecting microscope with a square grid (10x). The area (square millimeters) of each lesion was measured, summed per stomach and used as a lesion score. The person measuring the lesions did not know the groups to which test drugs were given. Tissue samples were then immersed into 10% formalin for histological observation, processed for routine light microscopy, sectioned at 5 µm, and stained with hematoxylin and eosin.
Determination of Acid Secretion
The experiment was performed using the same chamber system described above, except that the animals were not pretreated with omeprazole (Takeuchi et al., 1994). The mucosa mounted on a chamber was superfused at a flow rate of 1 ml/min with saline that was suffused with 100% O2, and kept in a reservoir. Acid secretion was measured by introducing an automatic titrator (Comtite-8; Hiranuma) into the perfusion system, and the titration was performed at luminal pH 7.0 using the pH-stat method and by adding 50 mM NaOH to the reservoir, in which both entry and exit tubes were positioned. After basal acid secretion had stabilized, the mucosa was exposed to 20 mM TC for 30 min. After application of TC, the mucosa was rinsed with saline, another 2 ml of saline was instilled, and the perfusion was resumed.
Determination of Prostaglandin E2
Gastric Mucosa. Mucosal PGE2 levels were measured in the stomach with or without TC treatment. In the former, animals without anesthesia were given indomethacin, SC-560, or rofecoxib orally and then killed 3 h later under deep ether anesthesia. In the latter, the chambered stomach of urethane-anesthetized animals was exposed to 20 mM TC plus 50 mM HCl for 30 min, and the mucosa was excised 60 min after the exposure. Indomethacin, SC-560, or rofecoxib was given intraduodenally 30 min before the TC treatment. In both studies, the corpus mucosa was isolated, weighed, and placed in a tube containing 100% ethanol plus 0.1 M indomethacin (Futaki et al., 1994). The samples were then minced with scissors, homogenized, and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant of each sample was used for determination of PGE2 by enzyme immunoassay using a PGE2 kit (Cayman Chemical, Ann Arbor, MI).
Carrageenan-Airpouch Model. Effects of various COX inhibitors on PGE2 content were examined in the exudates of a carrageenan-airpouch model. An airpouch was induced as described in detail previously (Edwards et al., 1981; Seibert et al., 1994, Barnett et al., 2000). In brief, 20 ml of air was injected subcutaneously on the back of the rat on the first day. Two days later, another 10 ml of air was injected at the same site. On the 5th day after the first injection, a further 10 ml of air was injected into the pouch. Twenty-four hours later, carrageenan [2 ml of a 1% (w/v) solution in saline] was injected into the airpouch. All of the injections were performed under light ether anesthesia. Six hours after the carrageenan injection, the rats were anesthetized with ether, and the pouch was carefully opened by making a small incision. Then, the exudate was collected and transferred to a tube. An aliquot of the exudates was frozen on dry ice and stored at -20°C for subsequent measurements of PGE2 concentration as described above. Indomethacin, SC-560, or rofecoxib was given orally 1 h before the last injection of carrageenan into the airpouch.
Analyses of COX-1 and COX-2 mRNAs by Reverse Transcription-Polymerase Chain Reaction
The stomachs were exposed to 20 mM TC plus 50 mM HCl for 30 min and quickly removed from the chamber at 30 or 90 min after the exposure. Each tissue was then frozen in liquid nitrogen and stored at -80°C until use. The tissue samples were pooled from two to three rats for extraction of total RNA, which was prepared by a single-step acid phenol-chloroform extraction procedure by use of TRIzol (Invitrogen, Carlsbad, CA). Total RNA primed by random hexadeoxy ribonucleotide was reverse-transcribed with the SuperScript preamplification system (Invitrogen). The sequences of sense and antisense primers for rat COX-1 and COX-2 as well as glyceraldehyde-3-phosphate dehydrogenase are referred to the previous studies (Bredt et al., 1991; Feng et al., 1993; Iso et al., 1995). An aliquot of the reverse transcription reaction product served as a template in 35 cycles of polymerase chain reaction with 1 min of denaturation at 94°C, 0.5 min of annealing at 58°C and 1 min of extension at 72°C on a thermal cycler. A portion of the polymerase chain reaction mixture was electrophoresed in 1.8% agarose gel in TAE buffer (40 mM Tris buffer, 2 mM EDTA, and 20 mM acetic acid, pH 8.1), and the gel was stained with ethidium bromide and photographed.
Preparation of Drugs
Drugs used in this study were urethane (Tokyo Kasei, Tokyo, Japan), taurocholate Na (Difco, Detroit, MI), indomethacin (Sigma-Aldrich, St. Louis, MO), SC-560 (Cayman Chemical), rofecoxib (synthesized by our group), carrageenan (Nacalai Tesque, Kyoto, Japan), and omeprazole (AstraZeneca, Möndal, Sweden). All COX inhibitors were suspended in a hydroxy propyl cellulose solution (Wako Pure Chemicals, Osaka, Japan). Omeprazole was suspended in a 0.5% carboxymethylcellulose solution. Other agents were dissolved in saline. Each agent was prepared immediately before use and administered i.p. or i.d. in a volume of 0.5 ml/100 g body weight, or applied topically to the chamber in a volume of 2 ml.
Statistics
Data are presented as the mean ± S.E. from four to six rats per group. Statistical analyses were performed using a two-tailed Dunnett's multiple comparison test, and values of P < 0.05 were regarded as significant.
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Results |
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Airpouch. Indomethacin given orally at 5 mg/kg significantly reduced exudate PGE2 content in the carrageenan-induced airpouch (Fig. 1B). Rofecoxib at 5 mg/kg also significantly decreased the exudate PGE2 content as effectively as indomethacin, whereas SC-560 at 5 mg/kg did not significantly affect the production of PGE2 in the carrageenan-induced airpouch model.
Because SC-560 and rofecoxib at 5 mg/kg significantly inhibited the production of PGE2 in the gastric mucosa and the airpouch exudate, respectively, these drugs at the dose were used in the subsequent studies as a selective COX-1 or COX-2 inhibitor.
Effects of Various COX Inhibitors on Gastric Functional Responses Induced by TC
PD Response. Under chambered conditions in the presence of omeprazole (inhibition of acid secretion) and exogenous acid (50 mM HCl plus 100 mM NaCl), a rat stomach generated a PD of -5968 mV (mucosa negative) and maintained a relatively constant GMBF during a 2-h experimental period. In control rats, the mucosal application of 20 mM TC plus 50 mM HCl for 30 min caused a marked reduction of PD from -63.3 ± 2.8 to -30.8 ± 2.6 mV, but after exposure the reduced PD was gradually normalized toward basal values, the degree of recovery being 49.6 ± 4.6% at 90 min after treatment (Fig. 2, A and B). Pretreatment of the animals with indomethacin, a nonselective COX-1 and COX-2 inhibitor, did not affect the reduction in PD caused by TC but significantly delayed the recovery of PD after exposure to TC, the degree of recovery being 12.4 ± 7.2% at 90 min posttreatment. Likewise, SC-560, a selective COX-1 inhibitor, also had no effect on PD reduction but significantly caused a delay in PD recovery, similar to indomethacin. In contrast, a selective COX-2 inhibitor rofecoxib did not have any effect on such PD responses after exposure to TC; the degree of PD recovery at 90 min post-treatment was 47.2 ± 10.9%, which was not statistically significant compared with control rats.
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GMBF Response. The GMBF significantly increased during exposure to 20 mM TC (plus 50 mM HCl), reaching a peak increase of 118.4 ± 27.4%, and remained significantly elevated for 30 min even after removal of TC from the chamber (Fig. 3). Even at 60 min after treatment, the GMBF showed a significant increase (35%) compared with preexposure values. This hyperemic response caused by TC was almost totally attenuated in rats pretreated with either indomethacin or SC-560, and in these animals the GMBF remained in similar ranges before and after exposure to TC, the peak increase being only 20% above basal levels. In contrast, rofecoxib had no effect on the gastric hyperemic response induced by TC, and the peak increase in GMBF was 84.3 ± 17.3%, which was not significantly different from that observed in control animals.
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Acid Back-Diffusion. When the stomach was exposed to 50 mM HCl in the absence of acid secretion induced by omeprazole, a small but significant loss of luminal H+ was consistently observed in control rats under normal conditions; acid loss (
H+) was less than 12 µEq/15 min. After the mucosal application of 20 mM TC, the loss of H+ was significantly increased, reaching a maximal value (23.8 ± 1.8 µEq/15 min) immediately after the exposure, then gradually decreasing to preexposure levels 120 min later (Fig. 4). Pretreatment with neither indomethacin, SC-560, nor rofecoxib had a significant effect on the increased mucosal permeability to H+ in response to TC; the magnitude of H+ observed immediately after TC treatment was 24.7 ± 2.0, 23.8 ± 2.1, and 19.8.5 ± 2.9 µEq/15 min, respectively.
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Acid Secretion. The chambered stomach secreted acid to keep the luminal pH at around 3.5 under urethane-anesthetized conditions. Mucosal application of 20 mM TC for 30 min reduced acid secretion by about 60% from 10.1 ± 1.8 to 3.8 ± 0.4 µEq/15 min (Fig. 5). This decrease in acid secretion induced by TC was significantly attenuated by previous administration of SC-560 as well as indomethacin, whereas rofecoxib had no effect on the reduced acid response to TC. In the group treated with indomethacin or SC-560, the acid secretion was also decreased by 20 to 30% after the mucosal exposure to TC, yet the changes were not statistically significant compared with the preexposure values. In the animals treated with rofecoxib, however, the acid secretion markedly decreased in response to TC, the degree of change being similar to that observed in the control group.
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Effects of Various COX Inhibitors on Mucosal Ulcerogenic Response Induced by TC
Mucosal application of 20 mM TC plus 50 mM HCl produced a few hemorrhagic lesions in the gastric mucosa of control rats, the lesion score being 3.6 ± 0.4 mm2 (Fig. 6). When animals were pretreated with indomethacin, the lesions induced by TC plus HCl were significantly aggravated; the lesion score was 19.2 ± 6.1 mm2, which was about 5 times greater than that in the control animals. Histologically, the stomachs of control rats only exhibited widespread exfoliation of epithelial cells without damage beyond the basement membrane, whereas in those pretreated with indomethacin the damage was deep into the mucosa with hemorrhagic changes (not shown). Likewise, SC-560 also significantly aggravated the mucosal ulcerogenic response to TC, the lesion score being 16.8 ± 2.8 mm2. In contrast, rofecoxib did not significantly affect the development of gastric lesions in response to TC, the lesion score being 5.1 ± 2.6 mm2.
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Effects of Various COX Inhibitors on Changes in Mucosal PGE2 Content Induced by TC
Mucosal exposure to 20 mM TC plus 50 mM HCl for 30 min stimulated PG biosynthesis to increase PGE2 content to about 10-fold the basal value (from 38.6 ± 4.6 to 685.2 ± 201.4 ng/g tissue) (Fig. 7). The PGE2 biosynthetic response induced by TC was totally blocked by previous i.d. administration of indomethacin. The increase in mucosal PGE2 was also significantly prevented by pretreatment with SC-560, the inhibition being 82.3%. In contrast, rofecoxib had no effect on the increased PG production after TC treatment.
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Expression of COX-1- and COX-2-mRNAs in the Stomach
Under normal conditions, only COX-1 gene expression was detected in the gastric mucosa and remained unchanged after exposure to 20 mM TC plus 50 mM HCl for 30 min (Fig. 8). On the other hand, the gene expression of COX-2 was negligible in the rat stomach, with or without TC treatment, when determined even at 90 min after the exposure.
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Discussion |
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First, we tested the activity of SC-560 or rofecoxib as a selective COX inhibitor. COX-1 is constitutively expressed in normal gastric mucosa and considered to generate PGs involved in the maintenance of essential physiological functions (Whittle, 1983; Simmons et al., 1991; Futaki et al., 1993). Mucosal PGE2 content in the normal stomach was significantly decreased by SC-560 but not rofecoxib. In contrast, rofecoxib but not SC-560 suppressed PGE2 production in a carrageenan-induced airpouch model. Because intrapleural injection of carrageenan produces an increase of PGE2 production and induction of de novo synthesis of COX-2 in pleural exudate cells (Masferrer et al., 1994; Nakatsugi et al., 1996), there is no doubt that this rofecoxib action is due to suppression of COX-2 activity. Thus, the present results confirmed that SC-560 and rofecoxib at the dose used, i.e., 5 mg/kg, selectively inhibits COX-1 and COX-2 activity, respectively.
It is known that the stomach responds to mucosal-damaging agents by altering various functions such as mucosal blood flow (Nobuhara and Takeuchi, 1984; Takeuchi et al., 1986, 1993). Although the role of PGs may vary with different types of mucosal irritants used to break the gastric mucosal barrier in the presence of luminal acid, this process is essentially mediated by endogenous PGs when the barrier is disrupted by TC (Nobuhara and Takeuchi, 1984; Takeuchi et al., 1986; Hirata et al., 1997). TC also damages surface epithelial cells and increases PG production in the gastric mucosa (Takeuchi et al., 1987, 1993), yet it remains unclear whether this PG response is mediated by the enzymatic activity of COX-1 or COX-2.
In the present study, we observed that the mucosal application of TC caused a reduction in PD followed by an increase in acid back-diffusion and GMBF, without extension to gross damage, consistent with previous findings of others and ourselves (Takeuchi et al., 1987, 1993). This treatment also enhanced PG biosynthesis in the stomach, inasmuch as the mucosal PGE2 content increased to 10-fold the basal value after exposure to TC. Whittle (1983) showed that gastric hyperemia after acid back-diffusion caused by TC is attenuated by indomethacin, suggesting the involvement of endogenous PGs in this phenomenon. As expected, we observed in this study that SC-560, a selective COX-1 inhibitor, as well as indomethacin attenuated the hyperemic response to TC, without affecting PD reduction and acid back-diffusion, resulting in aggravation of gastric lesions after TC treatment. In contrast, the selective COX-2 inhibitor rofecoxib had no effect on the gastric hyperemic response after barrier disruption by TC and did not cause any gross damage in the stomach. Furthermore, SC-560 but not rofecoxib significantly decreased the increase in the mucosal PGE2 content induced by TC, suggesting that the enhanced PG production after barrier disruption is associated with COX-1 activity. These findings also indicate that the gastric hyperemic response after acid back-diffusion is mediated by PGs and is dependent on COX-1 enzymatic activity. It is known that the gastric mucosa responds to damaging agents by decreasing acid secretion with a concomitant increase of mucosal blood flow (Nobuhara and Takeuchi, 1984; Takeuchi et al., 1986, 1987). In the present study, we also confirmed that mucosal exposure to 20 mM TC caused surface cell damage as represented by a reduction in PD, followed by a decrease of acid secretion. The reduced acid response in the stomach exposed to TC was significantly reverted by both indomethacin and SC-560, consistent with the present finding that endogenous PGs produced by COX-1 are involved in the GMBF response in the stomach after barrier disruption. As expected, rofecoxib did not have any effect on the reduced acid response in the stomach after exposure to TC, again confirming no room for COX-2 in the functional responses under such conditions. It is assumed that a decrease in acid secretion results in luminal alkalinization, which then contributes to maintenance of the microclimate for cellular restitution after barrier disruption (Svanes et al., 1982; Takeuchi et al., 1986). Certainly, other mediators such as nitric oxide and sensory neurons are also involved in the regulatory mechanism of gastric functional alterations after barrier disruption (Holzer et al., 1991; Takeuchi et al., 1993, 1994). However, because these factors interact with one another (Whittle et al., 1990), it is assumed that the lack of any one factor leads to failure of the full expression of these functional responses. We have previously shown that prednisolone, which is known to inhibit phospholipase A2 activity, also inhibited functional changes in the stomach induced by hyperosmolar NaCl (Nobuhara et al., 1985). In any case, the present study together with previous findings suggest that local application of mild irritants such as TC first releases arachidonic acid from membrane phospholipids in association with the mucosal irritation and then increases PG production due to COX-1 activity, resulting in functional alterations to enhance the mucosal resistance to acid injury.
The COX-1 protein is ubiquitously expressed in various tissues in the gastrointestinal tract, including the stomach, whereas the expression of COX-2 protein is absent from most gastrointestinal tissues (Kargman et al., 1993). We also confirmed that COX-1 mRNA was expressed in the gastric mucosa, irrespective of whether the stomach was exposed to TC. In contrast, the expression of COX-2 mRNA was not detected in the normal mucosa, and after exposure to TC remained undetectable for at least 90 min. As shown in the present study, the functional alterations were observed most markedly during or immediately after exposure to TC and subsided gradually after exposure, supporting the idea that COX-2 does not contribute to such responses occurring acutely in the stomach after barrier disruption. Certainly, the present study is acute in nature, and the results obtained will not be adequately applied in the stomach exposed to more prolonged irritation. Indeed, Barnett et al. (2000) reported the COX-2 expression in the stomach irritated with daily administration of 0.1% iodoacetamide for 5 days. We have previously reported that orally administered TC (20 mM plus 50 mM HCl) caused COX-2 induction in the stomach at 3 h after administration and showed adaptive gastric protection mediated by endogenous PGs derived from both COX-1 and COX-2 (Yamamoto et al., 1999). It is also known that COX-2 plays a crucial role in the mechanism of ulcer healing (Mizuno et al., 1997; Araki et al., 2002). Furthermore, Wallace et al. (2000) reported that inhibition of both COX-1 and COX-2 is required for the gastric ulcerogenic properties of NSAIDs. The same is true for the intestinal ulcerogenic properties of NSAIDs (Tanaka et al., 2002). We also reported that selective COX-2 inhibitors alone provoke gastric lesions in rats with adjuvant-induced arthritis (Kato et al., 2002). Thus, it is assumed that both COX-1 and COX-2 play roles in maintaining the integrity of the gastrointestinal mucosa, yet the importance of these COX isozymes varies depending on the experimental conditions.
Given the above-mentioned findings, the present study clearly showed that the COX-1 isozyme is involved in gastric functional responses, such as an increase of GMBF and a decrease in acid secretion, observed acutely after barrier disruption in the stomach. These functional alterations after barrier disruption are adaptive responses of the stomach and play an important role in protecting the mucosa against acid injury by disposing of H+ and maintaining a microclimate for cellular restitution. Because the stomach is continuously exposed to a variety of noxious stimuli such as acid, bile acids, and food-related chemicals, it seems that endogenous PGs produced by COX-1 are crucial in maintaining adaptive functional responses of the stomach under adverse conditions.
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Footnotes |
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ABBREVIATIONS: PG, prostaglandin; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; GMBF, gastric mucosal blood flow; TC, taurocholate; PD, potential difference; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole.
Address correspondence to: Dr. Koji Takeuchi, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan. E-mail: takeuchi{at}mb.kyoto-phu.ac.jp
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