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Mechanism of action of apomorphine on rat gastric secretion
European Journal of Pharmacology, 116 (1985) 279-285 Elsevier
279
M E C H A N I S M O F A C T I O N O F A P O M O R P H I N E O N RAT G A S T R I C S E C R E T I O N BRENDA COSTALL, ROBERT J. NAYLOR * and CONNIE C.W. TAN Postgraduate School of Studies in Pharmacology, University of Bradford, Bradford BD7 1DP, West Yorkshire, England
Received 10 April 1985, revised MS received 17 July 1985, accepted 23 July 1985
B. COSTALL, R.J. NAYLOR and C.C.W. TAN, Mechanism of action of apomorphine on gastric secretion, European J. Pharmacol. 116 (1985) 279-285. The effects of apomorphine on the volume of gastric secretion and its content of H +, K +, Na + and C1- were determined in conscious rats having gastric cannulas. Apomorphine dose-dependently (0.25-0.5 mg/kg s.c.) decreased the volume of gastric secretion, its acid concentration and, at the nighest dose, C1- concentration. However, Na + and K + concentrations were unchanged. The a 1- and aE-adrenoceptor antagonists prazosin (0.25-0.5 mg/kg i.p.) and yohimbine (5-10 mg/kg i.p.), the ill- and flE-adrenoceptor antagonist propranolol (5 mg/kg i.p.), the fl2-adrenoceptor antagonist ICI 118551 (1 mg/kg i.p.) and the dopamine receptor antagonists haloperidol (0.25-2.5 mg/kg i.p.), metoclopramide (2.5-10 mg/kg i.p.) or domperidone (2.5 mg/kg i.p.), administered alone, had little or no effect on the volume, H +, Na + or C1- concentrations of gastric secretion. Propranolol prevented the action of apomorpnine to reduce the volume of gastric secretion but failed to modify the reductions in H + and Cl- concentrations. The action of propranolol was mimicked by ICI 118551 but not by the fl~-adrenoceptor antagonist atenolol. Yonimbine, prazosin or domperidone had little or no effect on the actions of apomorphine. Haloperidol (0.5 mg/kg i.p.) and metoclopramide (10 mg/kg i.p.) antagonised apomorpnine's action to reduce H + and C1- concentrations but significantly enhanced the action of apomorpnine to reduce the volume of gastric secretion. The results suggest that the ability of apomorphine to reduce the volume of gastric secretion is mediated via fl2-adrenoceptors whilst acid concentration is reduced via an action on neuroleptic sensitive receptors. Apomorphine
fl2-Adrenoceptors
Gastric secretion
1. Introduction An established literature reports that sympathomimetic drugs can modify gastric secretion (see reviews by Holton, 1973; Sanders, 1976). Secretion can be modified by agents stimulating eq-, a 2- or fl-adrenoceptors, the direction of change being dependent on the in vivo or in vitro models used (see Canfield and Price, 1981) and by dopamine which is generally reported to decrease gastric secretion (Valenzuela and Grossman, 1976; Caldara et al., 1978; Hovendal and Bech, 1982), although an increased secretion can occur, dependent on the dose of dopamine (Hovendal and Bech, 1982). However, the particular role of dopamine receptors in the modulation of gastric * To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Neuroleptic-sensitive receptors
secretion is not clear since the effects of dopamine are reported to be modified both by the dopamine antagonist domperidone (Hovendal and Bech, 1982) and by the a- and fl-adrenoceptor antagonists phentolamine, propranolol and practolol (Hovendal and Bech, 1982). The present study investigates the actions of apomorphine to modify gastric secretion in the rat in an attempt to clarify the role of dopamine receptors in the control of gastric secretion.
2. Materials and methods 2.1. Measurement of gastric secretion
Male Sprague-Dawley (C.D.) rats weighing 325375 g were implanted with chronically indwelling
280 stainless steel gastric cannulas (7 mm diameter) exteriorised from the fundus of the stomach (chloral hydrate anaesthesia 300 m g / k g i.p.). A postoperative period of at least 14 days was allowed. Animals were held in individual perspex cages to which they had previously been acclimatised to allow sample collection with a minimum of stress. Before each experiment rats were fasted for 18 h, but allowed water ad libitum. Stomachs were rinsed with 6 x 5 ml warm normal saline followed by 3 x 5 ml warm distilled water and a polyethylene collecting tube attached to a graduated glass collecting vial was inserted into the cannula. The sample of gastric secretion collected in the first 15 min period was discarded but samples were then collected for analysis at hourly intervals for 4 h. Samples were centrifuged and the volume of fluid secreted during each period was determined by subtracting the sediment volume. A 0.1 ml aliquot of each sample, appropriately diluted with distilled water, was used for the determination of sodium and potassium (Corning 400, Flame Photometer) and a further 0.1 ml aliquot, diluted with distilled water neutralised with sodium bicarbonate was used for the potentiometric measurement of chloride (Selectrode F1012C1, Reference Electrode K701, Radiometer) and for subsequent auto titration with 0.01 N N a O H to an endpoint of pH 7.4 (ION85 Ion Analyser and ABu80 Autoburette TTA80 Titration Assembly, Electrode GK24Q1C, Radiometer) for the determination of titratable acid concentration and calculation of acid output.
2.2. Experimental design Rats received potential antagonists (prazosin, yohimbine, atenolol ICI 118551 [erythro-dl-l-(7methylindan-4-yloxy)-3-isopropylaminobutan-2-ol], dl-propranolol, metoclopramide, haloperidol or domperidone by the i.p. route) or vehicle 30 min before the collection of the first 15 min sample of gastric secretion which was discarded. Animals were then treated with apomorphine or vehicle (given s.c.) and returned to the individual perspex holding cages for sampling at hourly intervals for 4 h, the duration of experimentation being dictated by the prolongation of the action of apomorphine by some antagonist drugs. Five
animals were used in each treatment group, some treatments being repeated, and the significance of difference between treatments determined by twoway ANOVA followed by Dunnett's test. Where it is stated that data 'are not significantly different', then statistical analyses show P > 0.05 for inter group comparisons.
2.3. Drugs Apomorphine-HC1 (Sigma) was dissolved in nitrogen bubbled distilled water containing 0.01% sodium metabisulphite. Atenolol • HC1 (I.C.I.), ICI 118551 (I.C.I.), prazosin. HC1 (Pfizer), yohimbine • HC1 (Sigma), dl-propranolol-HC1 (I.C.I.) or metoclopramide dihydrochloride monohydrate (Beechams) was dissolved in distilled water and haloperidol or domperidone (Janssen) in the minimum quantity of lactic acid or glacial acetic acid respectively prepared to volume with distilled water neutralised with sodium bicarbonate. Doses were calculated as the base and administered in a volume of 1 ml/kg. 3. Results
3.1. Effects of apomorphine on secretion The volume of gastric secretion was significantly reduced by apomorphine, 0.35 and 0.5 m g / k g s.c., by 66 and 71% respectively during the 1 h period following administration; the mean value was also lower ( - 2 3 % ) following administration of 0.25 m g / k g s.c. apomorphine, but this failed to achieve significance. Values had returned to control levels by 2 h and there was no evidence of a rebound effect after 3 or 4 h; indeed, at 4 h the tendency was always towards a slight reduction. The acid concentration of gastric secretion was significantly reduced 1 h after apomorphine administration (by 53 and 68% respectively at 0.35 and 0.5 mg/kg) with a return to control values by 2 h. A significant reduction in CI- was only effected at 1 h by the highest dose of apomorphine. Na + and K ÷ concentrations of the gastric secretion were not altered following apomorphine treatment (fig. 1). The collection of gastric samples was not dis-
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Fig. 1. Effect of • • 0.25, • • 0.35 or • • 0.5 m g / k g s.c. a p o m o r p h i n e (n = 5) on N a ÷, K ÷, H ÷ or C1c o n c e n t r a t i o n s a n d v o l u m e of gastric secretion of the rat 1-4 h afler a d m i n i s t r a t i o n . Values are the m e a n responses, vertical bars show S.E.M. Significant reductions from control values (vehicle O . . . . . . © ) (n = 10) are i n d i c a t e d as * P < 0.01, ** P < 0.001.
rupted by the stereotyped sniffing with occasional biting which developed during the 1 h period following treatment with 0.5 m g / k g s.c. apomorphine. However, the use of higher doses (> 1 m g / k g s.c.) was prohibited by the development of intense stereotypic biting which impeded the collection of gastric samples. 3.2. Effects of prazosin, propranolol, atenolol, ICI 118551, yohimbine or neuroleptic agents on gastric secretion
Initial experiments (n = 5) established that prazosin (0.25 and 0.5 m g / k g i.p.), propranolol (5 m g / k g i.p.), atenolol (1 mg/kg i.p.), ICI 118551 (1 m g / k g i.p.), yohimbine (5 and 10 mg/kg i.p.), haloperidol (0.25, 0.5 and 2.5 m g / k g i.p.), metoclopramide (2.5, 5 and 10 mg/kg i.p.) or domperidone (2.5 m g / k g i.p.) had little or no action to modify the volume, H ÷, Na ÷, K ÷ or C1concentrations of gastric secretion over a 4 h period.
3. 3. Effects of catecholamine receptor antagonists on the apomorphine-induced changes in gastric secretion
Pretreatment with propranolol (5 m g / k g i.p.) prevented apomorphine's action to reduce the volume of gastric secretion but had little or no action on the reductions in H + concentration caused by apomorphine (fig~ 2). The action of propranolol on secretory volume, significant at l h, was mimicked by ICI 118551 (1 m g / k g i.p.) but not by atenolol (1 m g / k g i.p.) (fig. 2). Indeed, the action of atenolol was to enhance the effects of apomorphine to reduce the volume of gastric secretion, although this only achieved significance at 3 h. Similarly to atenolol, the action of yohimbine (10 mg/kg i.p.) and, in particular, prazosin (0.5 mg/kg i.p.) was to enhance rather than to reduce the action of apomorphine on the volume of gastric secretion and this achieved significance at 1 and 2 h following the administration of
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prazosin (fig. 3). Yohimbine, prazosin, ICI 118551 or atenolol had little or no consistent action to modify the ability of apomorphine to reduce H + concentration (figs. 2 and 3). Pretreatments with haloperidol (0.5 m g / k g i.p.) or metoclopramide (10 m g / k g i.p.) failed to antagonise the reduction in volume of gastric secretion caused by apomorphine; indeed, these two agents significantly enhanced this action of apomorphine response is indicated as * P < 0.001 and significant increase in its response as ÷ P < 0.05.
283
for a 3 h period (fig. 4). However, both haloperidol and metoclopramide were shown to antagonise apomorphine's action to reduce the H ÷ concentration in gastric secretion (fig. 4). Domperidone (2.5 m g / k g i.p.) had little or no effect on apomorphine's action on volume or H ÷ concentrations (fig. 4). The interaction between the catecholamine receptor antagonists and apomorphine failed to modify Na ÷ and K ÷ concentrations in gastric secretion. The changes of C1- concentration followed the changes in H ÷ concentration caused by the antagonist-apomorphine interaction.
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In the present study apomorphine was shown to consistently reduce both the volume and acid concentration of gastric secretion in rats having chronically indwelling gastric cannulas. However, whilst apomorphine is a recognised reference dopamine agonist (see review by Neumeyer et al., 1981), the actions on gastric secretion were shown to incorporate other mechanisms. The ability of apomorphine to reduce the secretory volume was antagonised by propranolol, indicating an action mediated via fl-adrenoceptors. The antagonism of this apomorphine response by the fl2-adrenoceptor antagonist ICI 118551 (Bilski et al., 1983), but not by the selective fll-adrenoceptor antagonist, atenolol (Barrett, 1977) suggests a selective fl2-adrenoceptor involvement. Whilst agonist action at fl-adrenoceptors in vivo is known to reduce secretory volume, an effect antagonised by propranolol (Misher et al., 1969; Yamaguchi et al., 1977), present and previous studies (see Yamaguchi et al., 1977) have failed to demonstrate an action of fl-adrenoceptor antagonists themselves (propranolol, ICI 118551 and atenolol) on gastric secretory volume which would not appear, therefore, to be under a tonic influence from fladrenoceptor mechanisms. Thus, a physiological relevance of the fl-adrenoceptors stimulated by
show S.E.M. Significant antagonism of the apomorphine response is indicated as * P < 0.05, ** P < 0.01 and significant increase in its response as ÷ P < 0.05, ÷ ÷ P < 0,01.
284 apomorphine (present studies) and isoprenaline (Misher et al., 1969; Yamaguchi et al., 1977) remains to be shown. Nevertheless, a pharmacological response in the stomach to apomorphine action on fl-adrenoceptors is not unique, for apomorphine can relax the muscle of isolated stomach strips by influencing fl-adrenoceptors (Costall et al., 1982). However, since isoprenaline and apomorphine exert an anti-secretory activity following intracerebroventricular injection (Yamaguchi et al., 1977; Costall et al., 1985), the site of drug action on fl-adrenoceptors to modify gastric secretory volume may involve a central effect. The failure of the a 1- and az-adrenoceptor antagonists, prazosin and yohimbine and the neuroleptic agents haloperidol, metoclopramide or domperidone to antagonise the apomorphine-induced reduction in secretory volume would further indicate a specific action of apomorphine on fl2adrenoceptors. Yet this is too simplistic an interpretation. Thus, the avadrenoceptor antagonist prazosin enhanced the inhibitory effect of apomorphine and the avadrenoceptor agonist phenylephrine has been shown to increase the volume of gastric secretion (Misher et al., 1969). Therefore, it is possible that a minor action of apomorphine on a~-adrenoceptors may oppose the dominant response to reduce secretory volume via fl2-adrenoceptor mechanisms. The effects of metoclopramide or haloperidol on apomorphine's ability to reduce the volume of gastric secretion are difficult to interpret. Firstly, there was no evidence that the neuroleptic agents would intensify the inhibitory action of apomorphine during the first hour, indicating an absence of a~-adrenoceptor (prazosin) antagonism. Secondly, the neuroleptic agents prolonged the apomorphine-volume effect for 3 h, some 2 h in excess of the known duration of action of apomorphine on systemic administration. This may indicate that during the 1-3 h period when apomorphine is administered alone a facilitatory action, which is neuroleptic sensitive, becomes effective to mask or oppose a less powerful inhibitory action which is neuroleptic-insensitive. The location and nature of this mechanism(s) is not known but the failure of domperidone to modify the action of apomorphine on secretory volume may indicate that apomor-
phine initiates its effects via central mechanisms, generally inaccessible to an agent such as domperidone which poorly penetrates the bloodbrain barrier (Costall et al., 1979). However, it is recognised that numerous hormonal mechanisms may exert a physiological a n d / o r pharmacological influence on gastric secretion, and these may also be relevant to the effects of apomorphine. This possibility remains to be investigated. Apomorphine reduced the acid concentration as well as the volume of gastric secretion. This effect on acid concentration was antagonised by haloperidol or metoclopramide but not by prazosin, propranolol, atenolol, ICI 118551 or yohimbine. Whilst such findings may indicate an important action on dopamine receptors, the failure of haloperidol or metoclopramide alone to modify basal acid concentration, noted in many previous studies (Meeroff, 1974; Jacoby and Brodie, 1967; Caldara et al., 1980), suggests the absence of a tonic inhibitory dopaminergic activity. Thus, the ability of apomorphine (present studies; see also Fjalland, 1974; Maeda-Hagiwara and Watanabe, 1983) or dopamine (Caldara et al., 1978) to reduce basal acid secretion may reflect a pharmacological intervention. Since dopamine fails to pass the bloodbrain barrier, the response to dopamine may indicate an action at peripheral sites. Apomorphine may have a similar site of action, but the apomorphine response was not antagonised by domperidone. Therefore, the dopamine receptor stimulated by apomorphine and blocked by haloperidol or metoclopramide may differ from other peripheral dopamine receptors, or the apomorphine effect to reduce acid concentration of gastric secretion may reflect a central action. The important conclusions of the study are two-fold. Whilst the mechanisms via which apomorphine can modify gastric secretion are clearly complex, it is suggested, firstly, that the reduction in volume of gastric secretion may be effected via/~2-adrenoceptors, whilst the reduction in acid concentration of gastric secretion may involve an action mediated at or via neuroleptic-sensitive receptors. It is emphasised that future studies should be designed to allow for this differentiation of effects on volume and acid concentration of gastric secretion.
285
Acknowledgements This work was supported by a research grant from the University of Bradford. The authors wish to thank Beechams, ICI, Janssen and Pfizer for gifts of drugs.
References Barrett, A.M., 1977, The pharmacology of atenolol, Postgrad. Med. J. 53, 52. Bilski, A.J., S.E. Halliday, J.D. Fitzgerald and J.L. Wale, 1983, The pharmacology of a fl2-selective adrenoceptor antagonist (ICI 118551), J. Cardiovasc. Pharmacol. 5, 430. Caldara, R., C. Ferrari, C. Barbieri and M. Romussi, 1980, Effect of four dopamine receptor antagonists on gastric secretion in healthy subjects, Scand. J. Gastroenterol. 15, 481. Caldara, R., C. Ferrari, M. Romussi, L. Bierti, S. Gandini and G. Curarelli, 1978, Effect of dopamine infusion on gastric and pancreatic secretion and on gastrin release in man, Gut 19, 724. Canfield, S.D. and C.A. Price, 1981, A comparison of the effects of sympathomimetic agents on gastric acid secretion by the rat stomach in vivo and in vitro, J. Physiol. 316, 11. Costall, B., D.H. Fortune and R.J. Naylor, 1979, Neuropharmacological studies on the neuroleptic potential of domperidone (R33812), J. Pharm. Pharmacol. 31, 344. Costall, B., S.J. Gunning and R.J. Naylor, 1982, Apomorphine contracts and relaxes circular smooth muscle of guinea pig stomach via action on adrenoceptor mechanisms, NaunynSchmiedeb. Arch. Pharmakol. 319, 226. Costall, B., R.J. Naylor, and C.C.W. Tan, 1985, A central site of dopamine agonist action to modify gastric secretion in the rat, European J. Pharmacol. 117, 61. Fjalland, B., 1974, Sympathomimetic amines and gastric secretion in rats, Acta Pharmacol. Toxicol. 35, 239.
Holton, P., 1973, Catecholamines and gastric secretion, in: The Pharmacology of Gastrointestinal Motility and Secretion, International Encyclopedia of Pharmacology and Therapeutics, ed. P. Holton (Pergamon Press, Oxford) section 39a. Hovendal, C.P. and K. Bech, 1982, Effect of dopamine on bethanechol-stimulated gastric mucosal blood flow and gastric acid secretion in dogs with gastric fistula, Scand. J. Gastroenterol. 17, 647. Jacoby, H.I. and D.A. Brodie, 1967, Gastrointestinal actions of metoclopramide, Gastroenterology 52, 676. Maeda-Hagiwara, M. and K. Watanabe, 1983, Influence of dopamine receptor agonists on gastric acid secretion induced by intraventricular administration of thyrotropin-releasing hormone in the perfused stomach of anaesthetised rats, Br. J. Pharmacol. 79, 297. Meeroff, J.C., 1974, The effect of metoclopramide on human gastric emptying and secretion, Acta Gastroenterol. Lat. Am. 6, 55. Misher, A., R.G. Pendelton and R. Staples, 1969, Effects of adrenergic drugs upon gastric secretion in rats, Gastroenterology 57, 294. Neumeyer, J.L., S. Lal and R.J. Baldessarini, 1981, Historical highlights of the chemistry, pharmacology and early clinical uses of apomorphine, in: Apomorphine and Other Dopaminomimetics, Vol. 1, eds. G.L Gessa and G. Umberto (Pergamon Press, New York) p. 1. Sanders, D.J., 1976, The agents and actions of sympathetic nerve and catecholamine inhibition of gastric mucosal function, Agents Actions 6, 385. Valenzuela, J.E. and M.I. Grossman, 1976, Dopamine stimulates pancreatic secretion and inhibits gastric secretion in dogs, Clin. Res. 24, 292A. Yamaguchi, I., J. Hiroi and S. Kumada, 1977, Central and peripheral adrenergic mechanisms regulating gastric secretion in the rat, J. Pharmacol. Exp. Ther. 203, 125.
279
M E C H A N I S M O F A C T I O N O F A P O M O R P H I N E O N RAT G A S T R I C S E C R E T I O N BRENDA COSTALL, ROBERT J. NAYLOR * and CONNIE C.W. TAN Postgraduate School of Studies in Pharmacology, University of Bradford, Bradford BD7 1DP, West Yorkshire, England
Received 10 April 1985, revised MS received 17 July 1985, accepted 23 July 1985
B. COSTALL, R.J. NAYLOR and C.C.W. TAN, Mechanism of action of apomorphine on gastric secretion, European J. Pharmacol. 116 (1985) 279-285. The effects of apomorphine on the volume of gastric secretion and its content of H +, K +, Na + and C1- were determined in conscious rats having gastric cannulas. Apomorphine dose-dependently (0.25-0.5 mg/kg s.c.) decreased the volume of gastric secretion, its acid concentration and, at the nighest dose, C1- concentration. However, Na + and K + concentrations were unchanged. The a 1- and aE-adrenoceptor antagonists prazosin (0.25-0.5 mg/kg i.p.) and yohimbine (5-10 mg/kg i.p.), the ill- and flE-adrenoceptor antagonist propranolol (5 mg/kg i.p.), the fl2-adrenoceptor antagonist ICI 118551 (1 mg/kg i.p.) and the dopamine receptor antagonists haloperidol (0.25-2.5 mg/kg i.p.), metoclopramide (2.5-10 mg/kg i.p.) or domperidone (2.5 mg/kg i.p.), administered alone, had little or no effect on the volume, H +, Na + or C1- concentrations of gastric secretion. Propranolol prevented the action of apomorpnine to reduce the volume of gastric secretion but failed to modify the reductions in H + and Cl- concentrations. The action of propranolol was mimicked by ICI 118551 but not by the fl~-adrenoceptor antagonist atenolol. Yonimbine, prazosin or domperidone had little or no effect on the actions of apomorphine. Haloperidol (0.5 mg/kg i.p.) and metoclopramide (10 mg/kg i.p.) antagonised apomorpnine's action to reduce H + and C1- concentrations but significantly enhanced the action of apomorpnine to reduce the volume of gastric secretion. The results suggest that the ability of apomorphine to reduce the volume of gastric secretion is mediated via fl2-adrenoceptors whilst acid concentration is reduced via an action on neuroleptic sensitive receptors. Apomorphine
fl2-Adrenoceptors
Gastric secretion
1. Introduction An established literature reports that sympathomimetic drugs can modify gastric secretion (see reviews by Holton, 1973; Sanders, 1976). Secretion can be modified by agents stimulating eq-, a 2- or fl-adrenoceptors, the direction of change being dependent on the in vivo or in vitro models used (see Canfield and Price, 1981) and by dopamine which is generally reported to decrease gastric secretion (Valenzuela and Grossman, 1976; Caldara et al., 1978; Hovendal and Bech, 1982), although an increased secretion can occur, dependent on the dose of dopamine (Hovendal and Bech, 1982). However, the particular role of dopamine receptors in the modulation of gastric * To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Neuroleptic-sensitive receptors
secretion is not clear since the effects of dopamine are reported to be modified both by the dopamine antagonist domperidone (Hovendal and Bech, 1982) and by the a- and fl-adrenoceptor antagonists phentolamine, propranolol and practolol (Hovendal and Bech, 1982). The present study investigates the actions of apomorphine to modify gastric secretion in the rat in an attempt to clarify the role of dopamine receptors in the control of gastric secretion.
2. Materials and methods 2.1. Measurement of gastric secretion
Male Sprague-Dawley (C.D.) rats weighing 325375 g were implanted with chronically indwelling
280 stainless steel gastric cannulas (7 mm diameter) exteriorised from the fundus of the stomach (chloral hydrate anaesthesia 300 m g / k g i.p.). A postoperative period of at least 14 days was allowed. Animals were held in individual perspex cages to which they had previously been acclimatised to allow sample collection with a minimum of stress. Before each experiment rats were fasted for 18 h, but allowed water ad libitum. Stomachs were rinsed with 6 x 5 ml warm normal saline followed by 3 x 5 ml warm distilled water and a polyethylene collecting tube attached to a graduated glass collecting vial was inserted into the cannula. The sample of gastric secretion collected in the first 15 min period was discarded but samples were then collected for analysis at hourly intervals for 4 h. Samples were centrifuged and the volume of fluid secreted during each period was determined by subtracting the sediment volume. A 0.1 ml aliquot of each sample, appropriately diluted with distilled water, was used for the determination of sodium and potassium (Corning 400, Flame Photometer) and a further 0.1 ml aliquot, diluted with distilled water neutralised with sodium bicarbonate was used for the potentiometric measurement of chloride (Selectrode F1012C1, Reference Electrode K701, Radiometer) and for subsequent auto titration with 0.01 N N a O H to an endpoint of pH 7.4 (ION85 Ion Analyser and ABu80 Autoburette TTA80 Titration Assembly, Electrode GK24Q1C, Radiometer) for the determination of titratable acid concentration and calculation of acid output.
2.2. Experimental design Rats received potential antagonists (prazosin, yohimbine, atenolol ICI 118551 [erythro-dl-l-(7methylindan-4-yloxy)-3-isopropylaminobutan-2-ol], dl-propranolol, metoclopramide, haloperidol or domperidone by the i.p. route) or vehicle 30 min before the collection of the first 15 min sample of gastric secretion which was discarded. Animals were then treated with apomorphine or vehicle (given s.c.) and returned to the individual perspex holding cages for sampling at hourly intervals for 4 h, the duration of experimentation being dictated by the prolongation of the action of apomorphine by some antagonist drugs. Five
animals were used in each treatment group, some treatments being repeated, and the significance of difference between treatments determined by twoway ANOVA followed by Dunnett's test. Where it is stated that data 'are not significantly different', then statistical analyses show P > 0.05 for inter group comparisons.
2.3. Drugs Apomorphine-HC1 (Sigma) was dissolved in nitrogen bubbled distilled water containing 0.01% sodium metabisulphite. Atenolol • HC1 (I.C.I.), ICI 118551 (I.C.I.), prazosin. HC1 (Pfizer), yohimbine • HC1 (Sigma), dl-propranolol-HC1 (I.C.I.) or metoclopramide dihydrochloride monohydrate (Beechams) was dissolved in distilled water and haloperidol or domperidone (Janssen) in the minimum quantity of lactic acid or glacial acetic acid respectively prepared to volume with distilled water neutralised with sodium bicarbonate. Doses were calculated as the base and administered in a volume of 1 ml/kg. 3. Results
3.1. Effects of apomorphine on secretion The volume of gastric secretion was significantly reduced by apomorphine, 0.35 and 0.5 m g / k g s.c., by 66 and 71% respectively during the 1 h period following administration; the mean value was also lower ( - 2 3 % ) following administration of 0.25 m g / k g s.c. apomorphine, but this failed to achieve significance. Values had returned to control levels by 2 h and there was no evidence of a rebound effect after 3 or 4 h; indeed, at 4 h the tendency was always towards a slight reduction. The acid concentration of gastric secretion was significantly reduced 1 h after apomorphine administration (by 53 and 68% respectively at 0.35 and 0.5 mg/kg) with a return to control values by 2 h. A significant reduction in CI- was only effected at 1 h by the highest dose of apomorphine. Na + and K ÷ concentrations of the gastric secretion were not altered following apomorphine treatment (fig. 1). The collection of gastric samples was not dis-
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rupted by the stereotyped sniffing with occasional biting which developed during the 1 h period following treatment with 0.5 m g / k g s.c. apomorphine. However, the use of higher doses (> 1 m g / k g s.c.) was prohibited by the development of intense stereotypic biting which impeded the collection of gastric samples. 3.2. Effects of prazosin, propranolol, atenolol, ICI 118551, yohimbine or neuroleptic agents on gastric secretion
Initial experiments (n = 5) established that prazosin (0.25 and 0.5 m g / k g i.p.), propranolol (5 m g / k g i.p.), atenolol (1 mg/kg i.p.), ICI 118551 (1 m g / k g i.p.), yohimbine (5 and 10 mg/kg i.p.), haloperidol (0.25, 0.5 and 2.5 m g / k g i.p.), metoclopramide (2.5, 5 and 10 mg/kg i.p.) or domperidone (2.5 m g / k g i.p.) had little or no action to modify the volume, H ÷, Na ÷, K ÷ or C1concentrations of gastric secretion over a 4 h period.
3. 3. Effects of catecholamine receptor antagonists on the apomorphine-induced changes in gastric secretion
Pretreatment with propranolol (5 m g / k g i.p.) prevented apomorphine's action to reduce the volume of gastric secretion but had little or no action on the reductions in H + concentration caused by apomorphine (fig~ 2). The action of propranolol on secretory volume, significant at l h, was mimicked by ICI 118551 (1 m g / k g i.p.) but not by atenolol (1 m g / k g i.p.) (fig. 2). Indeed, the action of atenolol was to enhance the effects of apomorphine to reduce the volume of gastric secretion, although this only achieved significance at 3 h. Similarly to atenolol, the action of yohimbine (10 mg/kg i.p.) and, in particular, prazosin (0.5 mg/kg i.p.) was to enhance rather than to reduce the action of apomorphine on the volume of gastric secretion and this achieved significance at 1 and 2 h following the administration of
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prazosin (fig. 3). Yohimbine, prazosin, ICI 118551 or atenolol had little or no consistent action to modify the ability of apomorphine to reduce H + concentration (figs. 2 and 3). Pretreatments with haloperidol (0.5 m g / k g i.p.) or metoclopramide (10 m g / k g i.p.) failed to antagonise the reduction in volume of gastric secretion caused by apomorphine; indeed, these two agents significantly enhanced this action of apomorphine response is indicated as * P < 0.001 and significant increase in its response as ÷ P < 0.05.
283
for a 3 h period (fig. 4). However, both haloperidol and metoclopramide were shown to antagonise apomorphine's action to reduce the H ÷ concentration in gastric secretion (fig. 4). Domperidone (2.5 m g / k g i.p.) had little or no effect on apomorphine's action on volume or H ÷ concentrations (fig. 4). The interaction between the catecholamine receptor antagonists and apomorphine failed to modify Na ÷ and K ÷ concentrations in gastric secretion. The changes of C1- concentration followed the changes in H ÷ concentration caused by the antagonist-apomorphine interaction.
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In the present study apomorphine was shown to consistently reduce both the volume and acid concentration of gastric secretion in rats having chronically indwelling gastric cannulas. However, whilst apomorphine is a recognised reference dopamine agonist (see review by Neumeyer et al., 1981), the actions on gastric secretion were shown to incorporate other mechanisms. The ability of apomorphine to reduce the secretory volume was antagonised by propranolol, indicating an action mediated via fl-adrenoceptors. The antagonism of this apomorphine response by the fl2-adrenoceptor antagonist ICI 118551 (Bilski et al., 1983), but not by the selective fll-adrenoceptor antagonist, atenolol (Barrett, 1977) suggests a selective fl2-adrenoceptor involvement. Whilst agonist action at fl-adrenoceptors in vivo is known to reduce secretory volume, an effect antagonised by propranolol (Misher et al., 1969; Yamaguchi et al., 1977), present and previous studies (see Yamaguchi et al., 1977) have failed to demonstrate an action of fl-adrenoceptor antagonists themselves (propranolol, ICI 118551 and atenolol) on gastric secretory volume which would not appear, therefore, to be under a tonic influence from fladrenoceptor mechanisms. Thus, a physiological relevance of the fl-adrenoceptors stimulated by
show S.E.M. Significant antagonism of the apomorphine response is indicated as * P < 0.05, ** P < 0.01 and significant increase in its response as ÷ P < 0.05, ÷ ÷ P < 0,01.
284 apomorphine (present studies) and isoprenaline (Misher et al., 1969; Yamaguchi et al., 1977) remains to be shown. Nevertheless, a pharmacological response in the stomach to apomorphine action on fl-adrenoceptors is not unique, for apomorphine can relax the muscle of isolated stomach strips by influencing fl-adrenoceptors (Costall et al., 1982). However, since isoprenaline and apomorphine exert an anti-secretory activity following intracerebroventricular injection (Yamaguchi et al., 1977; Costall et al., 1985), the site of drug action on fl-adrenoceptors to modify gastric secretory volume may involve a central effect. The failure of the a 1- and az-adrenoceptor antagonists, prazosin and yohimbine and the neuroleptic agents haloperidol, metoclopramide or domperidone to antagonise the apomorphine-induced reduction in secretory volume would further indicate a specific action of apomorphine on fl2adrenoceptors. Yet this is too simplistic an interpretation. Thus, the avadrenoceptor antagonist prazosin enhanced the inhibitory effect of apomorphine and the avadrenoceptor agonist phenylephrine has been shown to increase the volume of gastric secretion (Misher et al., 1969). Therefore, it is possible that a minor action of apomorphine on a~-adrenoceptors may oppose the dominant response to reduce secretory volume via fl2-adrenoceptor mechanisms. The effects of metoclopramide or haloperidol on apomorphine's ability to reduce the volume of gastric secretion are difficult to interpret. Firstly, there was no evidence that the neuroleptic agents would intensify the inhibitory action of apomorphine during the first hour, indicating an absence of a~-adrenoceptor (prazosin) antagonism. Secondly, the neuroleptic agents prolonged the apomorphine-volume effect for 3 h, some 2 h in excess of the known duration of action of apomorphine on systemic administration. This may indicate that during the 1-3 h period when apomorphine is administered alone a facilitatory action, which is neuroleptic sensitive, becomes effective to mask or oppose a less powerful inhibitory action which is neuroleptic-insensitive. The location and nature of this mechanism(s) is not known but the failure of domperidone to modify the action of apomorphine on secretory volume may indicate that apomor-
phine initiates its effects via central mechanisms, generally inaccessible to an agent such as domperidone which poorly penetrates the bloodbrain barrier (Costall et al., 1979). However, it is recognised that numerous hormonal mechanisms may exert a physiological a n d / o r pharmacological influence on gastric secretion, and these may also be relevant to the effects of apomorphine. This possibility remains to be investigated. Apomorphine reduced the acid concentration as well as the volume of gastric secretion. This effect on acid concentration was antagonised by haloperidol or metoclopramide but not by prazosin, propranolol, atenolol, ICI 118551 or yohimbine. Whilst such findings may indicate an important action on dopamine receptors, the failure of haloperidol or metoclopramide alone to modify basal acid concentration, noted in many previous studies (Meeroff, 1974; Jacoby and Brodie, 1967; Caldara et al., 1980), suggests the absence of a tonic inhibitory dopaminergic activity. Thus, the ability of apomorphine (present studies; see also Fjalland, 1974; Maeda-Hagiwara and Watanabe, 1983) or dopamine (Caldara et al., 1978) to reduce basal acid secretion may reflect a pharmacological intervention. Since dopamine fails to pass the bloodbrain barrier, the response to dopamine may indicate an action at peripheral sites. Apomorphine may have a similar site of action, but the apomorphine response was not antagonised by domperidone. Therefore, the dopamine receptor stimulated by apomorphine and blocked by haloperidol or metoclopramide may differ from other peripheral dopamine receptors, or the apomorphine effect to reduce acid concentration of gastric secretion may reflect a central action. The important conclusions of the study are two-fold. Whilst the mechanisms via which apomorphine can modify gastric secretion are clearly complex, it is suggested, firstly, that the reduction in volume of gastric secretion may be effected via/~2-adrenoceptors, whilst the reduction in acid concentration of gastric secretion may involve an action mediated at or via neuroleptic-sensitive receptors. It is emphasised that future studies should be designed to allow for this differentiation of effects on volume and acid concentration of gastric secretion.
285
Acknowledgements This work was supported by a research grant from the University of Bradford. The authors wish to thank Beechams, ICI, Janssen and Pfizer for gifts of drugs.
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Holton, P., 1973, Catecholamines and gastric secretion, in: The Pharmacology of Gastrointestinal Motility and Secretion, International Encyclopedia of Pharmacology and Therapeutics, ed. P. Holton (Pergamon Press, Oxford) section 39a. Hovendal, C.P. and K. Bech, 1982, Effect of dopamine on bethanechol-stimulated gastric mucosal blood flow and gastric acid secretion in dogs with gastric fistula, Scand. J. Gastroenterol. 17, 647. Jacoby, H.I. and D.A. Brodie, 1967, Gastrointestinal actions of metoclopramide, Gastroenterology 52, 676. Maeda-Hagiwara, M. and K. Watanabe, 1983, Influence of dopamine receptor agonists on gastric acid secretion induced by intraventricular administration of thyrotropin-releasing hormone in the perfused stomach of anaesthetised rats, Br. J. Pharmacol. 79, 297. Meeroff, J.C., 1974, The effect of metoclopramide on human gastric emptying and secretion, Acta Gastroenterol. Lat. Am. 6, 55. Misher, A., R.G. Pendelton and R. Staples, 1969, Effects of adrenergic drugs upon gastric secretion in rats, Gastroenterology 57, 294. Neumeyer, J.L., S. Lal and R.J. Baldessarini, 1981, Historical highlights of the chemistry, pharmacology and early clinical uses of apomorphine, in: Apomorphine and Other Dopaminomimetics, Vol. 1, eds. G.L Gessa and G. Umberto (Pergamon Press, New York) p. 1. Sanders, D.J., 1976, The agents and actions of sympathetic nerve and catecholamine inhibition of gastric mucosal function, Agents Actions 6, 385. Valenzuela, J.E. and M.I. Grossman, 1976, Dopamine stimulates pancreatic secretion and inhibits gastric secretion in dogs, Clin. Res. 24, 292A. Yamaguchi, I., J. Hiroi and S. Kumada, 1977, Central and peripheral adrenergic mechanisms regulating gastric secretion in the rat, J. Pharmacol. Exp. Ther. 203, 125.