Gastric acid stimulating action of cysteamine in the rat

European Journal of Pharmacology, 36 (1976) 331--336

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GASTRIC ACID STIMULATING ACTION OF CYSTEAMINE IN THE R A T YASUO ISHII, YUICHI F U J I I and MASAKO HOMMA

Research Laboratories, Nippon Kayaku Co., Shimo, Tokyo 115, Japan Received 15 July 1975, revised MS received 28 November 1975, accepted 3 December 1975

Y. ISHII, Y. F UJ II and M. HOMMA, Gastric a'cid stimulating action of cysteamine in the rat, European J. Pharmacol. 36 (1976) 331--336. Cysteamine was previously found to produce duodenal ulcers in rats. In the present study, the effect of cysteamine on gastric secretion was studied. Cysteamine administered by a parenterat or intraduodenal route induced a marked and sustained increase in gastric secretion in conscious and in anesthetized rats. The response to cysteamine or its metabolite, cystamine, appeared gradually and the maximum response was observed in 2 or 3 hr after the administration. The response to cysteamine was completely inhibited by vagotomy and inhibited dose-dependently by atropine methylbromide or hexamethonium. Cysteamine stimulated gastric acid secretion in conscious rats with chronic gastric fistula but not in rats with denervated gastric pouch. Tissue histamine levels in the gastric mucosa, abdominal muscle and whole blood were not affected by cysteamine. These results suggest that a cholinergic mechanism is involved in the gastric secretion induced by cysteamine.

Stimulation of gastric secretion

Cysteamine

1. Introduction Selye and Szabo (1973) reported an experimental model of acute duodenal ulcers in rats by a single administration of cysteamine • HC1 (~-mercaptoethylamine hydrochloride). In a previous study, influences of various factors and drugs on cysteamine-induced duodenal ulcers were investigated, and the o p t i m u m conditions for the production of duodenal ulcers were established (Fujii and Ishii, 1975). In this study, we found that atropine inhibited the duodenal ulcers in a dose-dependent manner, and pyloric ligation immediately prior to cysteamine dosing completely inhibited the duodenal ulcers and aggravated the ulcers in the stomach. These results show that the presence of gastric juice in the d u o d e n u m is a sine qua non for the production of duode-

Ulcerogenic agent

Cholinergic mechanism

nal ulcers induced by cysteamine in rats. Robert et al. (1974) presented similar results, b u t they observed that cysteamine inhibited gastric secretion in pylorus-ligated rats. Fletcher et al. (1959) also reported that cysteamine inhibited gastric secretion in the same preparation. Since pyloric ligation causes gastric hypersecretion in conscious rats (Brodie, 1966; Ishii, 1969), pylorus-ligated rats as originally described are unsuitable for a study of the effects of secretagogues such as histamine (Baume et al., 1965). Recently, Groves et al. (1974) reported that cysteamine stimulated gastric acid secretion in 1-hr pylorus-ligated rats which were treated with the drug 6 hr prior to the ligation. The present study is an attempt to ascertain the effect of cysteamine on gastric secretion in rats by means of different methods.

332 2. Materials and methods

2.1. Acute fistula rats Male Wistar rats weighing 200--250 g were used. After overnight fasting, they were anesthetized with urethane (1.2 g/kg s.c.) and an acute fistula was fixed in the forestomach as previously reported (Ishii and Shinozaki, 1968). The gastric juice was washed out with 10 ml of saline every hour and the washing solution was titrated with 0.1 N NaOH using phenolphtalein as indicator. Gastric acid output was expressed in pEq of HC1 secreted per hour.

2.2. Chronic fistula rats Male Wistar rats weighing 250--300 g were used. Two preparations were used: total gastric fistula and denervated gastric pouch, made following the techniques described by Komarov et al. (1963) and Alphin and Lin (1959), respectively, with slight modifications. The animals were fed commercial rat pellets and water ad libitum and were used for experiments at least no earlier than 1 week after the operation. Food was withdrawn overnight before the experiment. To collect gastric juice, a 2-ml hollow glass globe was connected to the gastric fistula with a silicone tube. By means of this method, gastric juice can be collected with the animals only slightly restrained.

2. 3. Analysis o f histamine in tissues The effect of cysteamine on histamine concentration in the mucosa of the stomach corpus, abdominal muscle and whole blood was investigated. The rats were sacrificed by bleeding from the common carotid arteries under ether anesthesia. The blood was heparinized and diluted with distilled water. After adding concentrated HCIO4, the mixture was shaken well and centrifuged at 0°C for 20 min at 8,000 × g and the supernatant was pipetted into a test tube. Other tissues were removed

Y. ISHII ET AL. quickly after sacrifice and frozen in liquid nitrogen. They were stored at --25°C but not kept for more than a week before use. The extraction and fluorometric analysis of histamine were performed, with slight modification, by the methods of Anton and Sayre (1969) and H~kanson et al. (1972).

2.4. Drugs The following drugs and chemicals were used: cysteamine and cysteamine hydrochloride (Tokyo-Kasei Kogyo), taurine (TokyoKasei Kogyo), cysteine (Tokyo-Kasei Kogyo), cystamine (Aldrich Chemical Co.), atropine methylbromide (C.H. Boehringer Sohn), hexamethonium bromide (Yamanouchi Seiyaku), histamine dihydrochloride (Tokyo-Kasei Kogyo), synthetic human gastrin I (Imperial Chemical Industry), and tetragastrin (t-amyloxycarbonyl Trp-Met-Asp-Phe-NH2, synthesized in our laboratory).

2.5. Statistics The data from all experiments are shown as mean and standard error. Comparison of differences between means was done using the Student's t-test.

3. Results

3.1. Effects in anesthetized rats Gastric acid secretory patterns induced by various routes of cysteamine dosing, and by an i.v. infusion of synthetic human gastrin (SHG) for 1 hr in anesthetized rats with acute fistula are shown in fig. 1. Secretory patterns of cysteamine and SHG were quite different. The maximum response to SHG appeared at the first hour after the beginning of infusion and the acid o u t p u t decreased rapidly after the withdrawal of infusion. On the contrary, the response to cysteamine appeared after some delay, the acid o u t p u t reaching a maximum in 2 or 3 hr after administration and

CYSTEAMINE ON GASTRIC SECRETION

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Fig. 1. Gastric acid secretion induced by cysteamine • HC1 and synthetic h u m a n gastrin I in anesthetized rats with a c u t e fistula. Cysteamine 100 mg/kg i.v. (©), i.d. (e), s.c. (A), i.m. (A); synthetic h u m a n gastrin I 20 p g / k g / h r i.v. (×). Significantly different from the initial value at **p = 0.01 or *p = 0.05.

decreasing gradually after 3 hr or more. The response to cysteamine given by the intraduodenal (i.d.) route was no less than when the drug was given by the i.v. route. Dose--response curves for cysteamine given by the i.m. and i.d. routes are shown in fig. 2. The response with the i.d. route was higher than

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Fig. 3. Effects of eysteamine-related compounds on gastric acid secretion in anesthetized rats with acute fistula. Cystamine 100 mg/kg i.v. (e), cysteine 100 mg/kg i.v. (o), e t h a n o l a m i n e 100 mg/kg i.v. (A), taurine 100 mg/kg i.v. (A). Significantly different from the initial value at **p = 0.01 or *p = 0.05.

that with the i.m. route. At a dose of 400 mg/kg i.m. or i.d., some animals died during the experiment from respiratory arrest probably due to the interaction of the drug and urethane. The effect of compounds structurally related to cysteamine, namely cystamine, taurine, cysteine and ethanolamine on gastric acid secretion was investigated (fig. 3). Among these, cystamine, an oxidized product of cysteamine, and taurine caused a sustained increase in gastric acid secretion at a dose of 100 mg/kg i.v. Ethanolamine elicited a temporary increase, but cysteine had no effect.

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The effect of atropine methylbromide (0.3 or 1.0 mg/kg, s.c.) and of cervical vagotomy on gastric acid secretion induced by 100 mg/kg i.d. of cysteamine in anesthetized rats is shown in fig. 4. Atropine inhibited the secretion in a dose-dependent manner, and cervical vagotomy completely abolished the response of cysteamine. As shown in fig. 5, a ganglion blocker, hexamethonium, also inhibited the cysteamine-induced gastric acid secretion in a dose-dependent manner.

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Fig. 4. Effects o f atrolSine m e t h y l b r o m i d e and cervical v a g o t o m y on cysteamine-induced gastric acid secretion in anesthetized rats with acute fistula. Bilateral cervical vagi were cut 1 hr before cysteamine. Cysteamine and atropine were given simultaneously. Cysteamine alone 100 mg/kg i.d. (o); cysteamine with a t r o p i n e 0.3 mg/kg s.c. (e), 1.0 mg/kg s.c. (A); cysteamine with v a g o t o m y (4). Significantly different f r o m the control value at **p = 0.01 or *p = 0.05.

3.3. Effects in conscious rats

In rats with a chronic gastric fistula, cysteamine produced a significant increase of gas-

Fig. 6. Effects of cysteamine and histamine on gastric acid secretion in conscious rats with gastric fistula. Cysteamine 200 mg/kg i.p. ($), 100 mg/kg i.p. (o); histamine 10 mg/kg i.m. (A). Significantly different from the initial value at **p = 0.01 or *p = 0.05.

tric acid secretion at a dose of 200 mg/kg i.p. with a 2 hr latency (fig. 6). Histamine (10 mg/kg i.m.), however, produced a maximum response at the first hour after the injection. The patterns of gastric volume secretion were approximately similar to those of acid secretion though the volume increases elicited by cysteamine were not significant owing to their large variances. On the contrary, in rats with a denervated gastric pouch, cysteamine (200 mg/kg i.p.) did not stimulate gastric acid or

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Fig. 5. Effect of h e x a m e t h o n i u m on cysteamine-induced gastric acid secretion in anesthetized rats with acute fistula. Cysteamine and h e x a m e t h o n i u m were given simultaneously. Cysteamine alone 100 mg/kg i.d. (o); cysteamine with h e x a m e t h o n i u m 30 mg/kg s.c. (e), 100 mg/kg s.c. (A). Significantly different from the control value at **P= 0.01 or *p -- 0.05.

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Fig. 7. Effects of cysteamine and tetragastrin on gastric acid and v o l u m e secretion in conscious rats with denervated gastric pouch. Cysteamine 200 mg/kg i.p. HC1 (o), v o l u m e (A); tetragastrin 500 pg/kg i.m. HC1 (e), v o l u m e (4). Significantly different from the initial value at **p = 0.01 or *p = 0.05.

CYSTEAMINE ON GASTRIC SECRETION secretion volume. In the same preparation, tetragastrin (500 tzg/kg i.m.) induced a marked increase in gastric secretion (fig. 7). 3.4. Effects on tissue histamine levels

Histamine levels in the mucosa of the stomach corpus, in abdominal muscle and in whole blood of rats sacrificed 3, 6 or 9 hr after administration of cysteamine (100 mg/kg i.m.) were compared with those in rats treated with saline alone. The histamine levels in each tissue at 3, 6 or 9 hr after dosing showed no significant difference in comparison with the level in control rats.

4. Discussion The present study showed that cysteamine given by various routes induced a marked and sustained increase in gastric acid secretion in conscious and anesthetized rats. The effect of cysteamine • HC1 on gastric secretion was not due to the hydrochloric acid moiety b u t to the drug. Regardless of the route of administration, the maximum response to cysteamine appeared 2 or 3 hr after administration, and the response lasted for about 5 hr. Cystamine, which is thought to be a metabolite of cysteamine, also caused a significant increase in gastric acid secretion. The reason for the delay and prolonged duration of action of cysteamine cannot be explained by the present investigation. The gastric secretion elicited by cysteamine was completely abolished by cervical vagotomy, and inhibited by atropine and hexamethonium in a dose-dependent manner. Cysteamine stimulated gastric acid secretion in conscious rats with chronic gastric fistula but did not stimulate the secretion in rats with a denervated gastric pouch. These results suggest that a cholinergic mechanism is involved in the gastric acid stimulating action of cysteamine. Groves et al. (1974) also observed that cysteamine stimulated gastric acid secretion of

335 long duration in rats and speculated that histamine might play a role in the effect. This hypothesis was based on the results reported by Mundy et al. (1963, 1967), Demaree et al. (1964) and Goncharenko and Briedis (1971). These authors had observed that cysteamine and cystamine caused histamine release, an increase in histidine decarboxylase activity, and a decrease in diamine oxydase activity. However, these studies were done independently of the gastric acid stimulating action of cysteamine. In the present investigation, cysteamine at a dose which stimulates gastric acid secretion did not affect histamine levels in the abdominal muscle, gastric mucosa and blood. These results suggest that cysteamine may not cause the secretion by systemic histamine release. Rosengren and Svensson (1969) proposed a model for the role of histamine in gastric acid secretion in which gastrin and vagally released acetylcholine directly stimulate histidine decarboxylase to produce histamine which initiates the secretion of acid. Therefore, further study is needed to ascertain the precise mechanism of cysteamine in gastric acid secretion. Cysteamine forms a part of the coenzyme A molecule and may be present in mammalian tissues in small amounts. Cysteamine was once used in experimental animals as a radioprotective and lathyrogenic substance. Recently, Prescott et al. (1974) used cysteamine in patients with paracetamol poisoning at a dose of 3.2 g i.v. over 20 hr. However, until Selye and Szabo (1973) reported the ulcerogenic action of cysteamine, there had been no report concerning the effect of cysteamine on gastric function or duodenal morphology. In a previous paper, we reported the effects of various factors and drugs on cysteamineinduced duodenal ulcer in rats, and suggested that the hypersecretion of gastric juice might be a sine qua non for the pathogenesis of ulceration. The present study ascertained the gastric acid stimulating action of cysteamine and supports the above hypothesis. Robert et al. (1970, 1971) reported methods for producing acute duodenal ulcers in rats by s.c.

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infusion for 48 hr of pentagastrin with histamine or carbachol, or by the constant infusion of the stomach and d u o d e n u m with acid. They showed that sustained gastric hypersecretion alone can produce ulcers in the duodenum of rats previously thought to be resistant to ulcerogenic drugs. Many studies have shown that there are excessive amounts of gastric juice in patients with duodenal ulcers. Therefore, it seems reasonable to explain the production of experimental duodenal ulcers by means of sustained gastric hypersecretion. Although the mechanism of gastric hypersecretion remains to be elucidated, cysteamine-induced duodenal ulceration in rats is an excellent model for the evaluation of antiulcer agents.

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