248
Agents and Actions vol. 9/3 (1979) BirkhS.user Verlag, Basel
The Effect of Cystamine on Gastric Secretion in the Rat by M.E. PARSONS, K.T. BUNCE, A. CURRINGTON, F. MATHESON and G. PIPKIN The Research Institute, Smith Kline & French Laboratories Ltd., Welwyn Garden City, Hertfordshire, England
Abstract The effects of cystamine on gastric secretion were studied in conscious and anaesthetized rat preparations. In the conscious gastric fistula rat cystamlne inhibited the basal acid output but increased pepsin output. This pepsinogogue action was inhibited by both atropine and metiamide. In the anaesthetized rat cystamine stimulated gastric acid output, an effect blocked by eimetldine which had an inhibitory E.D.50 which was not significantly different from that obtained against histamine-stimulated secretion in this preparation. Atropine at high doses failed to inhibit the response. Depletion of mast cell histamine by compound 48/80 and the removal of endogenous gastrln stores did not abolish the secretory response to eystamine. In the light of these results possible mechanisms of action for the secretagogue effects of cystamine are discussed.
Introduction A wide variety of ulcerogenic models have been developed in experimental animals over the past years. In most cases gastric ulceration has been studied and there have been relatively few models relating to duodenal ulcer. Recently however several groups of workers have reported that administration of cysteamine (fl-mercaptoethylamine) to rats causes duodenal ulcers [1-3]. The mechanism underlying the ulceration is not clear although both cysteamine and its metabolite cystamine (fl, fl'-diaminodiethyl disulphide) have been shown by some workers to stimulate gastric secretion in the rat [4]. However, other workers have reported inhibition of gastric secretion by cysteamine [2]. The studies described in the present paper were designed to establish the effect of cystamine on gastric secretion in the rat and to throw light on its mechanism of action. Cystamine was chosen since, in initial studies, cysteamine was found to cause toxic reactions in the conscious rat.
Methods The conscious rat The surgical preparation and post-operative care of gastric fistula rats has been described previously [5]. Female rats of the Wistar strain weighing approximately 200-250 g were used. The rats were prepared according to the method of LANE et al. [6]. A post-operative recovery period of at least two weeks was allowed. Before each experiment the rats were fasted for 18 h, but allowed water ad libitum. Each rat was lightly anaesthetized using halothane and a hypodermic needle inserted into a tail vein. The needle was con nected to a motor-driven syringe for intravenous infusion which was given at a rate of 3 ml/h. While still under anaesthesia the plug was removed from the gastric cannula and the stomach rinsed out with warm 0.9% saline. The rats were then placed in restraining cages and allowed to regain consciousness. Gastric juice samples were continuously collected by drainage and sampled for 6 h at hourly intervals. Acid output (/tmol/h) was determined by titration against 0.1 N NaOH to pH 7.4 using an automatic titrator (Radiometer type ABU 13). Pepsin output (pepsin units x 10-3/h) was determined by a modification of the autoanalyser method of VATIER et al. [7] using haemoglobin as the substrate. An intravenous infusion of 0.9% saline was given for the first hour of each experiment. The drugs were then added to this infusion as required. In the control experiments on basal secretion an intravenous infusion of 0.9% saline was given for the whole 6 h period.
The anaesthetized rat The preparation was a modification of the stomach perfusion method of GHOSH and SCmLD [8] described by PARSONS [9]. The vagotomized stomach of the starved, urethane-anaesthetized rat was perfused via cannulae placed in the oesophagus and in the pyloric antrum. The perfusate was collected via a funnel placed in the non-secretory rumen. Perfusion was carried out with 5.4% w/v glucose at 37~ and the perfusate was passed through a micro flow-type electrode system. Changes in hydrogen ion activity were recorded continuously on a potentiometric pen recorder. Drugs Halothane (Fluothane | I.C.I. Ltd.), atropine sulphate (B.D.H. Ltd.), Compound 48/80 (Burroughs Wellcome Ltd.), cystamine dihydrochloride (Sigma Chemical Co.).
249
The Effect of Cystamine on Gastric Secretion in the Rat
Metiamide and cimetidine were synthesized in our own laboratories.
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Analysis of the results Results are expressed as the mean + standard error of the mean (S.E.M.). The difference between two means was examined statistically using Student's 'C-test for unpaired data. A p value of less than 0.05 was considered to be significant,
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Results The conscious gastric fistula rat
Acid secretion. After a 1 h basal control period cystamine was infused for 5 h at two dose levels, 0.5 and 1.0 mg/kg/min i.v. in separate experiments. At the lower dose cystamine had no significant effect on acid output, but the dose of 1.0 mg/kg/min i.v. produced a significant (p < 0.05) inhibition of basal secretion (n = 12 in both cases), Figure 1. Peak inhibition was 41% and occurred 4 h after the commencement of the cystamine infusion. The inhibition was the result of a reduction of the volume of the juice secreted, the hydrogen ion concentration being unaffected. Pepsin secretion. In contrast, cystamine at a dose of 1 mg/kg/min i.v. produced a marked increase in pepsin output (Fig. 2). After a 1 h delay pepsin output increased from a mean of 6.62 units • 10-3/h to 30.54 units x 10-3/h, approximately a five-fold increase over basal. Since, as noted above, the volume of gastric juice was reduced in response to cystamine, this increase in pepsin output represents a large increase in pepsin concentration. The increase in pepsin output was maintained throughout the period of cystamine administration. A dose of 0.5 mg/kg/min had no significant effect on pepsin output. Antagonism of the pepsin response to cystamine by metiamide and atropine. In these studies an increase in pepsin output in response to an infusion of cystamine 1 mg/kg/min i.v. was established. Two hours after commencing cystamine administration, an infusion of metiamide (0.5 /zrnol/kg/min i.v.) or atropine (4 nmol/kg/ min i.v.) was started and the response followed over the next 3 h. Both antagonists caused a significant (p < 0.001) inhibition of the increase in pepsin output stimulated by cystamine (Fig. 3). The anaesthetized rat
The effect ofcystamine. Since the high basal secretion found in the conscious fistula rat can complicate interpretation of the action of secreta-
o HOURS
Figure1
Basal acid secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (11 n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( q n = 12). The arrow denotes the duration of the cystamine infusion. Means and standard errors of the mean (S.E.M.) are shown.
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Figure 2 Pepsin secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (11 n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( 0 n = 12). The arrow denotes the duration of the cystamine infusion. Means and standard errors of the mean (S.E.M.) are shown.
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Figure 3 Pepsin secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (m n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( ~ n - 12). At the arrow intravenous infusions of metiamide, 0.5/zmol/kg/min (V n = 12), or atropine, 4 nmol/kg/min ( O n = 12), were commenced. Means and standard errors of the mean (S.E.M.) are shown.
250
The Effect of Cystamine on Gastric Secretion in the Rat
gogues and secretory inhibitors [5], further studies were performed in the anaesthetized, vagotomized rat in which basal secretion is very low. Cystamine given by rapid intravenous injection stimulated gastric acid secretion in this preparation. A dose of 50 mg/kg produced a mean peak increase in H + output over basal of 3.34 + 0.51 (mean + S.E.M.) /~eq/min (n = 8). The pattern of acid secretion was markedly different from that obtained to other secretagogues in that the peak effect was not obtained until between 1 and 189 h after rapid intravenous injection and secretion was maintained for up to 5 h. In addition the secretory response was frequently biphasic, an initial rapid response being followed by a fall in secretion and then the secondary larger and prolonged response occurred. Inhibition by cimetidine Cimetidine given by rapid intravenous injection produced a dose-related inhibition of the secretory response to intravenous cystamine. Figure 4 shows that a dose of 2 r cimetidine almost completely abolished the response to 50 mg/kg cystamine. From a series of such experiments using graded doses of cimetidine an intravenous inhibitory E.D.50 of 0.75 /2mol/kg was calculated (n = 12, Fig. 5). The effect of atropine Atropine at a dose of 1 mg/kg i.v. failed to significantly inhibit cystamine-stimulated gastric secretion in this preparation (n = 6). The i.v. inhibitory E.D.~0 for atropine against carbacholstimulated secretion in this anaesthetized rat preparation is 1 ~tg/kg (Blakemore and Parsons, unpublished observations).
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Figure 5 The inhibitory effect of graded doses of cimetidine against cystamine-stimulated acid secretion in the anaesthetized rat. Each point shows the mean and standard error of the mean from four observations.
Studies in the histamine depleted rat Since the possibility existed that cystamine acts via a release of endogenous histamine, studies were carried out in the anaesthetized rat in which depletion of mast cell histamine had been induced by repeated administration of compound 48/80 at a dose of 0.1 mg/kg i.v. This dose of 48/80 produced an acid secretory response which exhibited rapid tachyphylaxis when the compound was repeatedly administered. When complete tachyphylaxis of the response had been achieved, a situation which presumably reflects depletion of mast cell histamine, cystamine given by rapid intravenous injection at a dose of 50 mg/kg produced its normal secretory response (n = 5). Studies in the antreetomized rat An alternative mechanism by which cystamine could stimulate gastric secretion is by release of endogenous gastrin. To investigate this possibility an acute antrectomy and duodenectomy was performed in the anaesthetized rat, thereby removing most of the sources of endogenous gastrin. Under these circumstances cystamine still produced its normal secretory response. Discussion
mln
Figure 4 Acid secretion in the anaesthetized rat. At A an intravenous injection of cystamine, 50 mg/kg, was administered. At B an intravenous injection of cimetidine, 2 ~ a o l / k g , was administered.
The first reports on the biological activity of cystamine described its effects on the cardiovascular system. In 1937 ROBBERS [10] showed that cystamine lowered blood pressure in the dog, an effect which he compared to the action of histamine. Subsequent studies both in vivo and in vitro showed that cystamine causes histamine release [11-13] and that the hypotensive effect is blocked by histamine HI-receptor antagonists [141.
251
The Effect of Cystamine on Gastric Secretion in the Rat
However, the effect of both cysteamine and cystamine on gastric acid secretion is unclear. Some workers have reported that these compounds stimulate gastric acid secretion in the rat [2, 3] whereas others have reported that cysteamine inhibits pre-existing basal acid secretion [2, 15]. The present results in the fistula rat indicate that cystamine inhibits the basal acid secretion and these results are in agreement with those of ROBERT et al. [2] and FLETCHER et al. [15] but in conflict with those of ISHII et al. [4] and GROVES et al. [3]. One possible explanation for this disagreement is differences in the rate of basal secretion existing in the preparations. In those studies in which stimulation of secretion was reported [3, 4] the level of basal secretion was low and in the studies of IsHn and co-workers [4] cystamine only increased secretion up to the basal levels seen in the present study. It may therefore be that, as suggested by other workers [3], a high basal secretion obscures the stimulant effect of these compounds. However, no explanation can be provided for the inhibitory effect of the high dose of cystamine. The possibility that a high basal secretion masks the stimulant effect of cystamine is further supported by the studies carried out in the anaesthetized vagotomized rat where the basal secretion is very low. Under these circumstances cystamine stimulates gastric acid secretion. However, the mechanism of this action of cystamine remains unclear. Isuu et al [4] found that the gastric secretion elicited by cysteamine was completely abolished by cervical vagotomy and was inhibited by atropine and hexamethonium in a dosedependent manner. These workers also found that cystamine stimulated gastric secretion in the conscious fistula rat but not in the rat fitted with a dennervated pouch. These results suggest an action via the vagus nerve. This however cannot be the sole mechanism of action of cystamine since the secretory response in our anaesthetized rat was obtained in the vagotomized preparation. In addition the failure of the high dose of atropine (some 1000 times the inhibitory E.D.50 against carbachol) to inhibit the secretion precludes a direct action via cholinergic receptors or via acetylcholine release. The fact that the specific histamine H 2receptor antagonist cimetidine inhibits the secretion and the inhibitory E.D.5o is not significantly different from that obtained against exogenously
administered histamine [16] suggests the involvement of a histaminergic pathway. As noted above, histamine release has been implicated in the effects of cystamine on the cardiovascular system. However, the present results using cystamine after compound 48/80 suggests that if cystamine does act on gastric secretion via a release of histamine, it is from stores which are not depleted by compound 48/80. One remaining possibility is that cystamine acts via a release of gastrin from the gastrointestinal tract since increased serum gastrin levels have been reported after cysteamine administration [ 17]. The antagonist studies described in the present paper do not argue against this hypothesis since we have shown cimetidine to inhibit pentagastrin-stimulated secretion in-the anaesthetized rat [16] whereas atropine even at high doses is ineffective against this stimulant [18]. However, the preliminary work in acutely antrectomized and duodenectomized rats suggests that gastrin release is not the mechanism of action, but further studies in chronic antrectomized animals are necessary. The results on pepsin secretion are in conflict with those reported previously [2, 15, 19] which showed that cysteamine inhibited pepsin secretion. The mechanism whereby, in our experiments, cystamine stimulates pepsin but not acid secretion is unclear since we have shown that histamine stimulated acid but not pepsin output in the conscious fistula rat and similarly pentagastrin is a good acid secretory stimulant (Bunce and Parsons, unpublished observations). There remains the possibility, in this preparation at least, of a cholinergic mechanism of action, a suggestion put forward by IsHn et al. [4]. The fact that both metiamide and atropine can inhibit the pepsigogue effect of cystamine does not help to clarify the mechanism of action since the reduction by the antagonists of the pre-existing basal secretion complicates interpretation of the results. In conclusion, the action of cystamine on gastric secretion is complex and depending on the experimental preparation either stimulation or inhibition of secretion can be observed. Although the most likely mechanism for its stimulant action is via release of endogenous histamine, other pathways cannot be excluded. It is also probable that the ulcerogenic effect of cystamine is a result, at least in part, of its gastric secretagogue action. Received 15 December 1978.
252
The Effect of Cystamine on Gastric Secretion in the Rat
References [1] H. SELVE and S. SZABO, Experimental Model for Production of Perforating Duodenal Ulcer by Cysteamine in the Rat, Nature 244, 458 (1973). [2] A. ROBERT, J.E. NEZAMIS, C. LANCASTER and J.N. BADALAMENTI,Cysteamine-lnduced Duodenal Ulcers: A New Model to Test Antiuleer Agents, Digestion i1, 199 (1974). [3] W.G. GROVES, J.H. SCHLOSSER and F.D. MEAD, Acid Hypersecretion and Duodenal Ulcers produced by Cysteamine in Rats, Res. Comm. Chem. Path. Pharmac. 9, 523 (1974). [4] Y. ISHII, Y. FuJtI and M. HOMMA, Gastric Acid Stimulating Action of Cysteamine in the Rat, Eur. J. Pharmac. 36, 331 (1976). [5] K.T. BUNCE and M.E. PARSONS, The Effect of Hexamethonium on Gastric Acid Secretion in the Conscious Rat, Agents and Actions 7, 507 (1977). [6] A. LANE, A.C. IvY and E.K. IvY, Response of the Chronic Gastric Fistula Rat to Histamine, Am. J. Physiol. 190, 221 (1957). [7] M.M. VATIER, A.M. CHERET and S. BONFmS, Le Dosage Automatique de l'Aetivitd Proteolytique du Suc Gastrique, Biol. Gastroenterol. 1, 15 (1968). [8] M.N. GHOSH and H.O. SCHILD, Continuous Recording of Acid Gastric Secretion in the Rat, Br. J. Pharmac. Chemother. 13, 54 (1958). [9] M.E. PARSONS, Quantitative Studies of Drug Induced Gastric Secretion, Ph.D. Thesis, University of London (1969). [10] H. ROBBERS, Die Pharmakologische Wirkung des Cystamins, einer Blutdrucksen-kenden Substanz, Arch. Exptl. Pathol. Pharmacol. 185, 461 (1937). [ 11 [ G.E. DEMAREE, R.L. MUNDY, M,H. HEIFFER and D.P. JACOBUS, In Vitro Histamine Release from Rat
Peritoneal Cells by fl-Mereaptoethylamine and Cystamine, J. Pharmac. exp. Ther. 144, 380 (1964). R.L. MUNDY, M.H. HEIFFER and G.E. DEMAREE, Histamine Release by fl-Mercaptoethylamine, Fedn. Proc. 22, 424 (1963). R.L. MUNDY, G.E. DEMAREE, D.P. JACOBUS and M.H. HEIEEER, fl-Mereaptoethylamine and CystamineInduced Histamine Release in the Dog, Arch. Int. Pharmacodyn. 165, 64 (1967). J. LECOMTE, Proprietes Pharmaeodynamiques de la Cystinamine, Arch. Int. Physiol. Biochem. 60, 179 (1952). T.L. FLETCHER, H.L. BUCHMAN, A.W. DAHL, J.E. JESSEPH and R.H. LARSEN, Decreased Gastric Secretory Activity Following Injection of Certain Maleimides, fl-Mercaptoethylamine, or the Combination of Both Types, J. Med. Chem. 1,275 (1959). R.W, BRIMBLECOMBE, W.A.M. DUNCAN, G.J. DURANT, J.C. EMMETT, C.R. GANELLIN and M.E. PARSONS, Cimetidine - a Non-Thiourea H2-Receptor Antagonist, J. Int. Med. Res. 312, 86 (1975). L.M. LICHTENBERGER,S. SZABO and J.S. TRIER,Duodenal Ulcerogens, Cysteamine and Propionitrile, Stimulate Serum Gastrin Levels in the Rat, Gastro enterology 73, 1305 (1977). A.R. HEDGES and M.E. PARSONS, The Effect of Anaesthesia on the Inhibition of Pentagastrin-Evoked Gastric Acid Secretion Induced by Atropine in the Rat, J. Physiol. 267, 181 (1977). L.E. BOREELA, K. SEETHALER and W. LIPPMANN, The Role of Gastric Acid and Pepsin Secretion in the Formation of Cysteamine-Induced Duodenal Ulcers in Rats, in: Prog. Peptic Ulcer (Proc. Conf. Exp. Ulcer) (Eds. G. Moznik and T. Javor; Akad. Kaido, Budapest, Hung.), p. 585.
[12] [13]
[14] [15]
[16]
[17]
[181
[19]
Agents and Actions vol. 9/3 (1979) BirkhS.user Verlag, Basel
The Effect of Cystamine on Gastric Secretion in the Rat by M.E. PARSONS, K.T. BUNCE, A. CURRINGTON, F. MATHESON and G. PIPKIN The Research Institute, Smith Kline & French Laboratories Ltd., Welwyn Garden City, Hertfordshire, England
Abstract The effects of cystamine on gastric secretion were studied in conscious and anaesthetized rat preparations. In the conscious gastric fistula rat cystamlne inhibited the basal acid output but increased pepsin output. This pepsinogogue action was inhibited by both atropine and metiamide. In the anaesthetized rat cystamine stimulated gastric acid output, an effect blocked by eimetldine which had an inhibitory E.D.50 which was not significantly different from that obtained against histamine-stimulated secretion in this preparation. Atropine at high doses failed to inhibit the response. Depletion of mast cell histamine by compound 48/80 and the removal of endogenous gastrln stores did not abolish the secretory response to eystamine. In the light of these results possible mechanisms of action for the secretagogue effects of cystamine are discussed.
Introduction A wide variety of ulcerogenic models have been developed in experimental animals over the past years. In most cases gastric ulceration has been studied and there have been relatively few models relating to duodenal ulcer. Recently however several groups of workers have reported that administration of cysteamine (fl-mercaptoethylamine) to rats causes duodenal ulcers [1-3]. The mechanism underlying the ulceration is not clear although both cysteamine and its metabolite cystamine (fl, fl'-diaminodiethyl disulphide) have been shown by some workers to stimulate gastric secretion in the rat [4]. However, other workers have reported inhibition of gastric secretion by cysteamine [2]. The studies described in the present paper were designed to establish the effect of cystamine on gastric secretion in the rat and to throw light on its mechanism of action. Cystamine was chosen since, in initial studies, cysteamine was found to cause toxic reactions in the conscious rat.
Methods The conscious rat The surgical preparation and post-operative care of gastric fistula rats has been described previously [5]. Female rats of the Wistar strain weighing approximately 200-250 g were used. The rats were prepared according to the method of LANE et al. [6]. A post-operative recovery period of at least two weeks was allowed. Before each experiment the rats were fasted for 18 h, but allowed water ad libitum. Each rat was lightly anaesthetized using halothane and a hypodermic needle inserted into a tail vein. The needle was con nected to a motor-driven syringe for intravenous infusion which was given at a rate of 3 ml/h. While still under anaesthesia the plug was removed from the gastric cannula and the stomach rinsed out with warm 0.9% saline. The rats were then placed in restraining cages and allowed to regain consciousness. Gastric juice samples were continuously collected by drainage and sampled for 6 h at hourly intervals. Acid output (/tmol/h) was determined by titration against 0.1 N NaOH to pH 7.4 using an automatic titrator (Radiometer type ABU 13). Pepsin output (pepsin units x 10-3/h) was determined by a modification of the autoanalyser method of VATIER et al. [7] using haemoglobin as the substrate. An intravenous infusion of 0.9% saline was given for the first hour of each experiment. The drugs were then added to this infusion as required. In the control experiments on basal secretion an intravenous infusion of 0.9% saline was given for the whole 6 h period.
The anaesthetized rat The preparation was a modification of the stomach perfusion method of GHOSH and SCmLD [8] described by PARSONS [9]. The vagotomized stomach of the starved, urethane-anaesthetized rat was perfused via cannulae placed in the oesophagus and in the pyloric antrum. The perfusate was collected via a funnel placed in the non-secretory rumen. Perfusion was carried out with 5.4% w/v glucose at 37~ and the perfusate was passed through a micro flow-type electrode system. Changes in hydrogen ion activity were recorded continuously on a potentiometric pen recorder. Drugs Halothane (Fluothane | I.C.I. Ltd.), atropine sulphate (B.D.H. Ltd.), Compound 48/80 (Burroughs Wellcome Ltd.), cystamine dihydrochloride (Sigma Chemical Co.).
249
The Effect of Cystamine on Gastric Secretion in the Rat
Metiamide and cimetidine were synthesized in our own laboratories.
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Analysis of the results Results are expressed as the mean + standard error of the mean (S.E.M.). The difference between two means was examined statistically using Student's 'C-test for unpaired data. A p value of less than 0.05 was considered to be significant,
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Results The conscious gastric fistula rat
Acid secretion. After a 1 h basal control period cystamine was infused for 5 h at two dose levels, 0.5 and 1.0 mg/kg/min i.v. in separate experiments. At the lower dose cystamine had no significant effect on acid output, but the dose of 1.0 mg/kg/min i.v. produced a significant (p < 0.05) inhibition of basal secretion (n = 12 in both cases), Figure 1. Peak inhibition was 41% and occurred 4 h after the commencement of the cystamine infusion. The inhibition was the result of a reduction of the volume of the juice secreted, the hydrogen ion concentration being unaffected. Pepsin secretion. In contrast, cystamine at a dose of 1 mg/kg/min i.v. produced a marked increase in pepsin output (Fig. 2). After a 1 h delay pepsin output increased from a mean of 6.62 units • 10-3/h to 30.54 units x 10-3/h, approximately a five-fold increase over basal. Since, as noted above, the volume of gastric juice was reduced in response to cystamine, this increase in pepsin output represents a large increase in pepsin concentration. The increase in pepsin output was maintained throughout the period of cystamine administration. A dose of 0.5 mg/kg/min had no significant effect on pepsin output. Antagonism of the pepsin response to cystamine by metiamide and atropine. In these studies an increase in pepsin output in response to an infusion of cystamine 1 mg/kg/min i.v. was established. Two hours after commencing cystamine administration, an infusion of metiamide (0.5 /zrnol/kg/min i.v.) or atropine (4 nmol/kg/ min i.v.) was started and the response followed over the next 3 h. Both antagonists caused a significant (p < 0.001) inhibition of the increase in pepsin output stimulated by cystamine (Fig. 3). The anaesthetized rat
The effect ofcystamine. Since the high basal secretion found in the conscious fistula rat can complicate interpretation of the action of secreta-
o HOURS
Figure1
Basal acid secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (11 n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( q n = 12). The arrow denotes the duration of the cystamine infusion. Means and standard errors of the mean (S.E.M.) are shown.
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0
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Figure 2 Pepsin secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (11 n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( 0 n = 12). The arrow denotes the duration of the cystamine infusion. Means and standard errors of the mean (S.E.M.) are shown.
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Figure 3 Pepsin secretion in gastric fistula rats during an i.v. infusion of 0.9% saline alone, 3 ml/h (m n = 12), and in the presence of cystamine, 1 mg/kg/min i.v. ( ~ n - 12). At the arrow intravenous infusions of metiamide, 0.5/zmol/kg/min (V n = 12), or atropine, 4 nmol/kg/min ( O n = 12), were commenced. Means and standard errors of the mean (S.E.M.) are shown.
250
The Effect of Cystamine on Gastric Secretion in the Rat
gogues and secretory inhibitors [5], further studies were performed in the anaesthetized, vagotomized rat in which basal secretion is very low. Cystamine given by rapid intravenous injection stimulated gastric acid secretion in this preparation. A dose of 50 mg/kg produced a mean peak increase in H + output over basal of 3.34 + 0.51 (mean + S.E.M.) /~eq/min (n = 8). The pattern of acid secretion was markedly different from that obtained to other secretagogues in that the peak effect was not obtained until between 1 and 189 h after rapid intravenous injection and secretion was maintained for up to 5 h. In addition the secretory response was frequently biphasic, an initial rapid response being followed by a fall in secretion and then the secondary larger and prolonged response occurred. Inhibition by cimetidine Cimetidine given by rapid intravenous injection produced a dose-related inhibition of the secretory response to intravenous cystamine. Figure 4 shows that a dose of 2 r cimetidine almost completely abolished the response to 50 mg/kg cystamine. From a series of such experiments using graded doses of cimetidine an intravenous inhibitory E.D.50 of 0.75 /2mol/kg was calculated (n = 12, Fig. 5). The effect of atropine Atropine at a dose of 1 mg/kg i.v. failed to significantly inhibit cystamine-stimulated gastric secretion in this preparation (n = 6). The i.v. inhibitory E.D.~0 for atropine against carbacholstimulated secretion in this anaesthetized rat preparation is 1 ~tg/kg (Blakemore and Parsons, unpublished observations).
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0.33 1 CIMETIDINE llmo/kg
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Figure 5 The inhibitory effect of graded doses of cimetidine against cystamine-stimulated acid secretion in the anaesthetized rat. Each point shows the mean and standard error of the mean from four observations.
Studies in the histamine depleted rat Since the possibility existed that cystamine acts via a release of endogenous histamine, studies were carried out in the anaesthetized rat in which depletion of mast cell histamine had been induced by repeated administration of compound 48/80 at a dose of 0.1 mg/kg i.v. This dose of 48/80 produced an acid secretory response which exhibited rapid tachyphylaxis when the compound was repeatedly administered. When complete tachyphylaxis of the response had been achieved, a situation which presumably reflects depletion of mast cell histamine, cystamine given by rapid intravenous injection at a dose of 50 mg/kg produced its normal secretory response (n = 5). Studies in the antreetomized rat An alternative mechanism by which cystamine could stimulate gastric secretion is by release of endogenous gastrin. To investigate this possibility an acute antrectomy and duodenectomy was performed in the anaesthetized rat, thereby removing most of the sources of endogenous gastrin. Under these circumstances cystamine still produced its normal secretory response. Discussion
mln
Figure 4 Acid secretion in the anaesthetized rat. At A an intravenous injection of cystamine, 50 mg/kg, was administered. At B an intravenous injection of cimetidine, 2 ~ a o l / k g , was administered.
The first reports on the biological activity of cystamine described its effects on the cardiovascular system. In 1937 ROBBERS [10] showed that cystamine lowered blood pressure in the dog, an effect which he compared to the action of histamine. Subsequent studies both in vivo and in vitro showed that cystamine causes histamine release [11-13] and that the hypotensive effect is blocked by histamine HI-receptor antagonists [141.
251
The Effect of Cystamine on Gastric Secretion in the Rat
However, the effect of both cysteamine and cystamine on gastric acid secretion is unclear. Some workers have reported that these compounds stimulate gastric acid secretion in the rat [2, 3] whereas others have reported that cysteamine inhibits pre-existing basal acid secretion [2, 15]. The present results in the fistula rat indicate that cystamine inhibits the basal acid secretion and these results are in agreement with those of ROBERT et al. [2] and FLETCHER et al. [15] but in conflict with those of ISHII et al. [4] and GROVES et al. [3]. One possible explanation for this disagreement is differences in the rate of basal secretion existing in the preparations. In those studies in which stimulation of secretion was reported [3, 4] the level of basal secretion was low and in the studies of IsHn and co-workers [4] cystamine only increased secretion up to the basal levels seen in the present study. It may therefore be that, as suggested by other workers [3], a high basal secretion obscures the stimulant effect of these compounds. However, no explanation can be provided for the inhibitory effect of the high dose of cystamine. The possibility that a high basal secretion masks the stimulant effect of cystamine is further supported by the studies carried out in the anaesthetized vagotomized rat where the basal secretion is very low. Under these circumstances cystamine stimulates gastric acid secretion. However, the mechanism of this action of cystamine remains unclear. Isuu et al [4] found that the gastric secretion elicited by cysteamine was completely abolished by cervical vagotomy and was inhibited by atropine and hexamethonium in a dosedependent manner. These workers also found that cystamine stimulated gastric secretion in the conscious fistula rat but not in the rat fitted with a dennervated pouch. These results suggest an action via the vagus nerve. This however cannot be the sole mechanism of action of cystamine since the secretory response in our anaesthetized rat was obtained in the vagotomized preparation. In addition the failure of the high dose of atropine (some 1000 times the inhibitory E.D.50 against carbachol) to inhibit the secretion precludes a direct action via cholinergic receptors or via acetylcholine release. The fact that the specific histamine H 2receptor antagonist cimetidine inhibits the secretion and the inhibitory E.D.5o is not significantly different from that obtained against exogenously
administered histamine [16] suggests the involvement of a histaminergic pathway. As noted above, histamine release has been implicated in the effects of cystamine on the cardiovascular system. However, the present results using cystamine after compound 48/80 suggests that if cystamine does act on gastric secretion via a release of histamine, it is from stores which are not depleted by compound 48/80. One remaining possibility is that cystamine acts via a release of gastrin from the gastrointestinal tract since increased serum gastrin levels have been reported after cysteamine administration [ 17]. The antagonist studies described in the present paper do not argue against this hypothesis since we have shown cimetidine to inhibit pentagastrin-stimulated secretion in-the anaesthetized rat [16] whereas atropine even at high doses is ineffective against this stimulant [18]. However, the preliminary work in acutely antrectomized and duodenectomized rats suggests that gastrin release is not the mechanism of action, but further studies in chronic antrectomized animals are necessary. The results on pepsin secretion are in conflict with those reported previously [2, 15, 19] which showed that cysteamine inhibited pepsin secretion. The mechanism whereby, in our experiments, cystamine stimulates pepsin but not acid secretion is unclear since we have shown that histamine stimulated acid but not pepsin output in the conscious fistula rat and similarly pentagastrin is a good acid secretory stimulant (Bunce and Parsons, unpublished observations). There remains the possibility, in this preparation at least, of a cholinergic mechanism of action, a suggestion put forward by IsHn et al. [4]. The fact that both metiamide and atropine can inhibit the pepsigogue effect of cystamine does not help to clarify the mechanism of action since the reduction by the antagonists of the pre-existing basal secretion complicates interpretation of the results. In conclusion, the action of cystamine on gastric secretion is complex and depending on the experimental preparation either stimulation or inhibition of secretion can be observed. Although the most likely mechanism for its stimulant action is via release of endogenous histamine, other pathways cannot be excluded. It is also probable that the ulcerogenic effect of cystamine is a result, at least in part, of its gastric secretagogue action. Received 15 December 1978.
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The Effect of Cystamine on Gastric Secretion in the Rat
References [1] H. SELVE and S. SZABO, Experimental Model for Production of Perforating Duodenal Ulcer by Cysteamine in the Rat, Nature 244, 458 (1973). [2] A. ROBERT, J.E. NEZAMIS, C. LANCASTER and J.N. BADALAMENTI,Cysteamine-lnduced Duodenal Ulcers: A New Model to Test Antiuleer Agents, Digestion i1, 199 (1974). [3] W.G. GROVES, J.H. SCHLOSSER and F.D. MEAD, Acid Hypersecretion and Duodenal Ulcers produced by Cysteamine in Rats, Res. Comm. Chem. Path. Pharmac. 9, 523 (1974). [4] Y. ISHII, Y. FuJtI and M. HOMMA, Gastric Acid Stimulating Action of Cysteamine in the Rat, Eur. J. Pharmac. 36, 331 (1976). [5] K.T. BUNCE and M.E. PARSONS, The Effect of Hexamethonium on Gastric Acid Secretion in the Conscious Rat, Agents and Actions 7, 507 (1977). [6] A. LANE, A.C. IvY and E.K. IvY, Response of the Chronic Gastric Fistula Rat to Histamine, Am. J. Physiol. 190, 221 (1957). [7] M.M. VATIER, A.M. CHERET and S. BONFmS, Le Dosage Automatique de l'Aetivitd Proteolytique du Suc Gastrique, Biol. Gastroenterol. 1, 15 (1968). [8] M.N. GHOSH and H.O. SCHILD, Continuous Recording of Acid Gastric Secretion in the Rat, Br. J. Pharmac. Chemother. 13, 54 (1958). [9] M.E. PARSONS, Quantitative Studies of Drug Induced Gastric Secretion, Ph.D. Thesis, University of London (1969). [10] H. ROBBERS, Die Pharmakologische Wirkung des Cystamins, einer Blutdrucksen-kenden Substanz, Arch. Exptl. Pathol. Pharmacol. 185, 461 (1937). [ 11 [ G.E. DEMAREE, R.L. MUNDY, M,H. HEIFFER and D.P. JACOBUS, In Vitro Histamine Release from Rat
Peritoneal Cells by fl-Mereaptoethylamine and Cystamine, J. Pharmac. exp. Ther. 144, 380 (1964). R.L. MUNDY, M.H. HEIFFER and G.E. DEMAREE, Histamine Release by fl-Mercaptoethylamine, Fedn. Proc. 22, 424 (1963). R.L. MUNDY, G.E. DEMAREE, D.P. JACOBUS and M.H. HEIEEER, fl-Mereaptoethylamine and CystamineInduced Histamine Release in the Dog, Arch. Int. Pharmacodyn. 165, 64 (1967). J. LECOMTE, Proprietes Pharmaeodynamiques de la Cystinamine, Arch. Int. Physiol. Biochem. 60, 179 (1952). T.L. FLETCHER, H.L. BUCHMAN, A.W. DAHL, J.E. JESSEPH and R.H. LARSEN, Decreased Gastric Secretory Activity Following Injection of Certain Maleimides, fl-Mercaptoethylamine, or the Combination of Both Types, J. Med. Chem. 1,275 (1959). R.W, BRIMBLECOMBE, W.A.M. DUNCAN, G.J. DURANT, J.C. EMMETT, C.R. GANELLIN and M.E. PARSONS, Cimetidine - a Non-Thiourea H2-Receptor Antagonist, J. Int. Med. Res. 312, 86 (1975). L.M. LICHTENBERGER,S. SZABO and J.S. TRIER,Duodenal Ulcerogens, Cysteamine and Propionitrile, Stimulate Serum Gastrin Levels in the Rat, Gastro enterology 73, 1305 (1977). A.R. HEDGES and M.E. PARSONS, The Effect of Anaesthesia on the Inhibition of Pentagastrin-Evoked Gastric Acid Secretion Induced by Atropine in the Rat, J. Physiol. 267, 181 (1977). L.E. BOREELA, K. SEETHALER and W. LIPPMANN, The Role of Gastric Acid and Pepsin Secretion in the Formation of Cysteamine-Induced Duodenal Ulcers in Rats, in: Prog. Peptic Ulcer (Proc. Conf. Exp. Ulcer) (Eds. G. Moznik and T. Javor; Akad. Kaido, Budapest, Hung.), p. 585.
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