Difference in Mode of Action of Cimetidine Ane Gefarnate on Endogenous Prostacyclin, Prostaglandin E2 and Thromboxane in Rat Gastric Mucosa
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Tohoku J. exp. Med., 1983, 140, 407-412 Difference in Mode of Action of Cimetidine ane Gefarnate on Endogenous Prostacyclin, Prostaglandin E2 and Thromboxane in Rat Gastric Mucosa TETSUO A.RAKAWA,HAJIME NAKAMURA,SHINJI CHONO HIROSHI SATOH, HIROAKI YAMADA, TOKIO ONO and KENZO KOBAYASHI The Third Department of Internal Medicine, Osaka City University Medical School, Osaka 545 ARAKAWA,T., NAKAMURA,H., CHONO,S., SATOH,H., YAMADA,H., ONo, T. and KoBAYASHI,K. Difference in Mode of Action of Cimetidine and Gefarnate on Endogenous Prostacyclin, Prostaglandin E2 and T hromboxane in Rat Gastric Mucosa. Tohoku J. exp. Med., 1983, 140 (4), 407-412 The effects of cimetidine and gefarnate on endogenous prostacyclin, prostaglandin E2 and thromboxane were studied in vivo in rat gastric mucosa. The animals received cimetidine (20 mg/kg, i.p.) and/or gefarnate (100 mg/kg, s.c.) twice a day for 7 days. Gastric mucosal 6-keto-prostaglandin F1 (as prostacyclin), prostaglandin E2 and thromboxane B2 (as thromboxane A2) were determined by radioimmunoassay. Cimetidine reduced prostacyclin, prostaglandin E2, but not thromboxane A2. Gefarnate inhibited the cimetidine-induced reduction of prostacyclin and prostaglandin E2; in cimetidine-untreated controls, it did not produce an increase in those prostaglandins and thromboxane A2 above the normal levels. - prostacyclin (PGI2); prostaglandin E2; cimetidine; gefarnate; gastric mucosa Cimetidine, an H2-receptor antagonist, has been shown to prevent several experimental ulcerations (Mann and Sachdev 1976; Safaie-Shirazi et al. 1976) and has been reported to be highly effective in the management of upper gastrointestinal hemorrhage (Dunn et al. 1978; Eden and Kern 1978) or peptic ulcers (Henn et al. 1975; Winship 1978; Bianchiporro et al. 1979). However, ulcers often recur within a few months after interruption of cimetidine treatment (Hanskey and Korman 1979; CURE 1980; Marks et al. 1981). Guslandi et al. (1978) found that cimetidine reduces the protective effect of gastric mucus. In contrast with cimetidine, prostaglandins (PGs) prevent gastrointestinal ulceration (Carter et al. 1974; Lipmann 1974) by mechanisms other than inhibition of acid secretion (Henn et al. 1975; Cano et al. 1976; Pounder et al. 1976; Richardson et al. 1976). This protection has been called "cytoprotection" (Robert 1976; Whittle 1976; Carmichael et al. 1977; Robert et al. 1979). Gefarnate, an isoprenoid whose anti-ulcer action is due to its protection of the gastric mucosa, has Receivedfor publication, December13, 1982. 407 408 T. Arakawaet al. recently been shown to inhibit the decrease of the gastric mucosal PGI2 and PGE2 levels in rats stressed by water-immersion (Kobayashi et al. 1983). The present study was undertaken to determine whether cimetidine could reduce endogenous PGs in rat gastric mucosa, and whether gefarnate could inhibit the cimetidine-induced reduction of endogenous PGs. MATERIALS AND METHODS Male Wistar rats weighing 180-220 g were used throughout this investigation. They were divided into four groups of 5 rats each and used for radioimmunoassay of gastric mucosal PGI2, PGE2 and TX. Group I received saline (i.p.) plus peanut oil (s.c.) ; Group II, saline (i.p.) plus gefarnate (s.c.) ; Group III, cimetidine (i.p.) plus peanut oil (s.c.) ; Group IV, cimetidine (i.p.) plus gefarnate (s.c.). The animals were injected twice a day for 7 days with cimetidine dissolved in saline (20 mg/kg) and/or gefarnate dissolved in peanut oil (100 mg/kg). They were fasted during 24 hr preceding the last injection and decapitated 1 hr thereafter. The stomach was quickly removed, opened along the greater curvature and washed with tap water. The gastric mucosa was separated from the muscle layer both in the fundic and antral portions by our freezed-splitting technique (Kobayashi et al. 1981). The gastric mucosal tissue thus obtained was weighed, 3H-PGE2 and 3H-6-keto-PGFi« (1000 cpm; New England Nuclear) were added as recovary markers and the tissue was homogenized with a glass homogenizes in 5 ml methanol, containing 3 X 10-4M sodium mecrofenamate (Waner-Lambert Co., Ltd.) to stop PG synthesis during subsequent procedures. The homogenizes was washed out with 10 ml chloroform, the resultant solution was mixed vigorously with homogenate and allowed to stand for 30 min at room temperature. PGs and TX were extracted as has been described (Kobayashi et al. 1983). The solution was filtrated and washed with Folch's solution (CHC13-CH3OH;2:1 by volume) and evaporated to dryness at 40°C under a nitrogen stream. The residue was dissolved in 5 ml CC14,10 ml of 10% methanol-phosphate buffer were added, followed by vigorous shaking and 10-min centrifugation at 2;,000 Xg. The upper phase was adjusted to pH 3 with 1 N HCI, 10 ml of ethylacetate were added and this was followed by vigorous shaking and 5-min centrifugation at 2000 X g. To the upper phase we added 200 ,ul of 1 N NH3OH and 2 ml methanol; this was followed by mixing and evaporating at 40°C under a nitrogen stream. The residue was subjected to thin-layer chromatography (TLC) using Analtech 0.25 mm silica gel G precoated plates to separate PGE-TX and 6-keto-PGFi« The organic phase of ethylacetate : acetic acid : isooctan : H2O (110: 20: 50: 100) was used for development (Sun et al. 1977). PGE2, 6-keto-PGFi«, and TXB2 were determined by radioimmunoassay using 3H-PGE2, 3H-6-keto-PGFi«, 3H-TXB2 (New England Nuclear) and antisera. PGE2-antiserum was obtained from Institut Pasteur Production; 6-keto- PGF«i- and TXB2-antisera were from Ono Pharmaceutical Co., Ltd. RESULTS Effect of cimetidine on gastric mucosal PGI2, PGE2 and T X In the gastric mucosa of control rats, the PG and TX levels were (mean±S.E.) PGI2, 3,814.7±370.2; PGE2, 3,123.7±269.5; TXA2, 73.2±55.6 ng/g tissue in fundic mucosa ; PGI2, 7,307.8±1,387.6; PGE2, 4,606.6±584.2; TXA2, 333.6±103.8 ng/g tissue in antral mucosa. The repeated i.p. administration of cimetidine reduced fundic PGI2 by about 45% (p<0.01) and antral PGI2 by 35% (not significant) ; fundic PGE2 was reduced by about 65% (p<0.01), antral PGE2 by 70% (p<0.01). TXA2 was slightly Antiulcer Agents and Mucosal Prostaglandins 409 TABLE 1. The gastric mucosal prostaglandin and thromboxane levels of controls, gefarnate-treated rats, cimetidine-treated rats, and cimetidine plus gefarnate-treated rats increased in the fundic and reduced in the antral mucosa, but these changes were not statistically significant (Table 1). Effect of gefarnate on gastric mucosal PGI2i PGE2 and TX As shown in Table 1, the repeated s.c. administration of gefarnate to otherwise untreated healthy rats failed to increase their mucosal PGI2, PGE2 and TXA2 levels. However, gefarnate significantly inhibited the cimetidine-induced reducton of fundic mucosal PGI2 (p<0.05) and PGE2 (p<0.025) and of antral PGE2 (p<0.05). DISCUSSION Cimetidine, introduced by Brimblecombe et al. (1975) as the third H2-receptor antagonist, exerts definite inhibition on acid secretion. In man, cimetidine is remarkably effective in the management of upper gastrointestinal hemorrhage (Eden and Kern 1978) and peptic ulcers (Henn et al. 1975; Winship 1978; Bianchiporro et al. 1979), although there is some doubt regarding its efficacy in gastric ulcer patients (Ciclitira et al. 1977; Freston 1978). In addition to its antisecretory effect, cimetidine may exert a cytoprotective effect. In rats, this agent reduced the incidence of ulcers induced with aspirin plus HCl (Bommelaer and Guth 1979). On the other hand, in similar experiments, Carmichael et al. (1978) noted no signi- ficant difference between cimetidine-treated and untreated rats, suggesting that the antiulcer effect of cimetidine may be solely due to its antisecretory action. Levine et al. (1979), who studied the role of cimetidine in the prevention of stress- induced ulcers, concluded that it may prevent those types of ulcers by inhibiting acid secretion only, because cimetidine was not effective in preventing the develop- ment of the ulcers in the presence of exogenous HCI. 410 T. Arakawa et al. Kenyon et al. (1977) demonstrated that cimetidine did not affect a gastric potential difference, nor did it affect the disruption of the mucosal barrier by sodium taurocholate in Heidenheim pouches of antrectomized dogs. On the other hand. Ivey et al. (1975) showed that cimetidine effected a rise in the gastric potential difference in man. While the inhibition of acid secretion and the consequent decrease in the luminal hydrogen ion concentration can increase the transmucosal potential difference, this does not imply a tightening of mucosal barriers (Read and Levin 1975). In the present study, the gastric mucosal PGI2 and PGE2 levels were reduced by cimetidine. This may explain the occurrence of ulcer relapses (Hanskey and Korman, 1979; CURE 1980; Marks et al. 1981; Martin et al. 1981) upon discon- tinuation of cimetidine because those PGs are potent effectors of gastric cyto- protection (Whittle 1976; Robert et al. 1979; Konturek et al. 1981). The cimetidine-induced reduction of the mucoprotective potential (Guslandi et al. 1978) may be due to reduction of mucosal PGI2 and PGE2. The mechanism by which cimetidine effects a decrease in PGI2 and PGE2 remains unknown. Robert et al. (1978) proposed that mild acid may stimulate mucosal PGs; our previous study, in which we determined mucosal PGE2 by radioimmunoassay (Arakawa et al. 1981), lent support to this hypothesis. We suggest that the observed action of cimetidine on the PGI2 and PGE2 levels may be secondary to its inhibition of acid secretion. We previously reported that gefarnate inhibited the decrease of the gastric mucosal PGI2 and PGE2 concentration in rats stressed by water-immersion (Koba- yashi et al.