- Email: [email protected]
Devazepide Attenuates dl-Fenfluramine-Induced Suppression of Gastric Emptying but Not Food Intake in the 17 h Food-Deprived Rat
Physiology & Behavior, Vol. 62, No. 3, pp. 545–550, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 / .00
PII S0031-9384(97)00015-2
Devazepide Attenuates dl-Fenfluramine-Induced Suppression of Gastric Emptying but Not Food Intake in the 17 h Food-Deprived Rat JOHN FRANCIS,* 1 COLIN T. DOURISH† AND STEVEN J. COOPER‡ *Centre for Human Nutrition, University of Sheffield, Northern General Hospital, Sheffield, S5 7AU, UK, †Cerebrus, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7PN, UK, and ‡Department of Psychology, University of Durham, Science Laboratories, South Road, Durham, DH1 3LE, UK Received 19 June 1995; Accepted 29 January 1997 FRANCIS, J., C. T. DOURISH, AND S. J. COOPER, Devazepide attenuates dl- fenfluramine-induced suppression of gastric emptying but not food intake in the 17 h food-deprived rat. PHYSIOL BEHAV 62(3) 545–550, 1997.—Recently a number of studies have provided evidence which suggests that CCK and 5-HT interact in the control of food intake. The present experiments further examine this mechanism and the possibility that CCK and 5-HT interact in the control of gastric emptying. The selective CCK-A receptor antagonist, devazepide, (0.03–3.0 mg/kg) administered alone had no intrinsic effect on gastric emptying. Devazepide (0.1 and 0.3 mg/kg) blocked dl-fenfluramine-induced (3.0 mg/kg) suppression of gastric emptying. However, devazepide (0.03–3.0 mg/kg) failed to attenuate the anorectic effect of the same dose of dl-fenfluramine. These results suggest that under the present experimental conditions CCK and 5-HT interact in the regulation of gastric emptying but not food intake. Thus the interaction between CCK and 5-HT in the regulation of gastric emptying appears not to affect the control of ingestive behaviour. q 1997 Elsevier Science Inc.
vazepide, reversed the anorectic effect of d -fenfluramine ( 4 ) . These data suggested that serotonergic influences on control of feeding may depend upon the mediation of CCK. Later, Cooper and Dourish also reported that devazepide attenuated the anorectic effect of exogenously administered 5-HT ( 5 ) . Clifton and Cooper extended this work and, using a meal pattern analysis, demonstrated that in freely-feeding rats devazepide antagonised the reduction in meal size produced by d fenfluramine ( 3 ) . This result was confirmed in later work by Grignaschi and colleagues ( 14 ) . Hence, the effect of d-fenfluramine to reduce meal size appears to be due, in part, to an action of CCK at CCK-A receptors. Evidence from electrophysiological studies also suggest that there are potential interactions between CCK and serotonin. Thus, Boden and colleagues recorded intracellularly from neurones in the dorsal raphe nucleus (2). Some of the neurones which were inhibited by 5-HT (a characteristic of serotonergic neurones) were excited by CCK-8 but not by the selective CCKB agonist, pentagastrin. Moreover, the excitatory effect of CCK8 was blocked by devazepide but not by the selective CCK-B receptor antagonist, L-365,260. Hence, CCK may act at CCK-A receptors to influence activity of 5-HT-containing neurones in the dorsal raphe. The anorectic effect of both cholecystokinin (CCK) and dl fenfluramine, an indirectly-acting serotonin (5-HT) agonist, may
RECENT studies have identified reciprocal interactions between cholecystokinin (CCK) and serotonin (5-HT) in their effects on food intake. Thus, Stallone and colleagues were the first to show that metergoline, a 5-HT receptor antagonist, attenuated the anorectic effect of CCK-8 in the 3 h food-deprived rat (23). Metergoline also reversed the anorectic effect of dl -fenfluramine, an effect which is believed to be mediated by central 5-HT 1 receptors (19). Furthermore, Stallone and colleagues showed that the anorectic effect of CCK-8 was not affected by the peripheral 5HT receptor antagonist, xylamidine. They proposed that exogenous CCK-8 acts peripherally, presumably to produce a vagallymediated signal, to alter central 5-HT activity and hence induce a satiety effect (23). Subsequently, Poeschla and colleagues and Grignaschi and colleagues confirmed the results reported by Stallone and colleagues (14,20,21). Inhibiting central 5-HT activity, through, for example, the action of selective 5-HT 1A agonist, would be expected to oppose the anorectic effect of exogenous CCK. Poeschla and colleagues reported that 8-OH-DPAT, 5HT 1A agonist, did attenuate the feeding-suppressant effect of exogenous CCK-8 (21). However, it should be noted that Ebenezer and Brooman failed to replicate this result (8). In contrast to the mediation of CCK-8-induced satiety via 5-HT mechanisms, there are data which indicate an interaction in the opposite direction. Thus, Cooper and colleagues demonstrated that the selective CCK-A receptor antagonist, de1
To whom requests for reprints should be addressed.
545 / eh11 5015 Mp 545 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
546
FRANCIS, DOURISH AND COOPER
depend, at least in part, on their inhibitory effect on gastric emptying (17,22). This apparent overlap in the effects of CCK and 5-HT on food intake and gastric emptying suggests that interactions may occur between these two transmitters to produce, in effect, a common mechanism underlying their effects. The aim of the first experiment, reported here, was to examine the possible effect of devazepide on the suppression of gastric emptying produced by dl -fenfluramine (10,21). If there is a mechanism in common, and there is some coupling between CCK- and 5-HT-dependent processes, respectively, then CCK receptor blockade may impede dl -fenfluramine’s effect. In addition, it was first necessary to establish under the present conditions the effects of devazepide administered alone on gastric emptying. The second study investigated whether devazepide would attenuate or block dl -fenfluramine-induced anorexia in food-deprived rats. EXPERIMENT ONE The effect of devazepide on dl-fenfluramine-induced suppression of gastric emptying in 17 h food-deprived rats. METHOD AND MATERIALS
Animals Seventy-four adult male Sprague–Dawley rats (Charles River, Manston, UK), weighing between 250–300 g at the time of testing, were used. They were housed individually with ad libitum access to food pellets (Diet 41B, Heygate and Sons, Northants, U.K.). They were maintained under a 12 h L:D cycle with lights on at 0800 h. Prior to testing animals were transferred to test cages, where they were food-deprived overnight (approx. 17 h) with ad lib access to water. Drugs dl-Fenfluramine was purchased from Sigma Chemical Co. Ltd. (Poole, UK). dl-fenfluramine was dissolved in isotonic saline. Devazepide was kindly donated by Merck, Sharp and Dohme (Harlow, UK). Devazepide was suspended in 0.5% carboxymethylcellulose. Drugs were administered in a volume of 1ml/kg (doses refer to base). Test Meal Carboxymethylcellulose (low viscosity, 10–20 cps, Sigma Chemical Co. Ltd., Poole, UK) was added in four 5-g portions (20 g in total) to 250 ml of distilled water. After the addition of each portion, the mixture was agitated in a blender for 1 min. Casein (bovine milk: Sigma Chemical Co. Ltd.) was added to this mixture in two 8-g portions (16 g in total), followed by 8 g of cane sugar (household; Tate and Lyle, London, UK) and 8 g cornstarch (Sigma Chemical Co. Ltd.). Following the addition of each portion of these ingredients, the mixture was agitated for 1 min. The resulting mixture was a white semisolid paste, which was placed in a refrigerator overnight to allow any air trapped in the mixture to escape. Two hours before use, the mixture was removed from the refrigerator and allowed to reach room temperature. Procedure To investigate the intrinsic effect of devazepide on gastric emptying, groups of rats ( n ¢ 6 per group ) were injected subcutaneously ( SC ) with devazepide ( 0.03, 0.1, 0.3, 1.0 and 3.0 mg / kg ) or vehicle ( carboxymethylcellulose ) 30 min prior to testing.
Previous experiments in our laboratories have shown that 3 mg/kg dl -fenfluramine significantly and reliably suppresses gastric emptying and food intake in 17h food-deprived rats, and therefore, this dose was used in the present studies (12). The doses of devazepide used had no intrinsic effect on gastric emptying and were chosen on the basis of the results of the first experiment. Thus, groups of rats, (n ¢ 5 per group) were injected (SC) 30 min before testing with devazepide (0.03, 0.1 and 0.3 mg/kg) or vehicle, while dl-fenfluramine (3 mg/kg) or vehicle (saline) were injected intraperitoneally 20 min before receiving the test meal. Following administration of drugs rats were given by gavage a weighed amount of test meal (approx. 3 g) equalling 3 ml in volume. Thirty min later, the rats were killed by cervical dislocation and laparotomized. The stomachs were ligated at the oesophagus and pylorus, to prevent leakage, removed and weighed. The stomachs were then opened and any remaining test meal was rinsed out. Any excess water was removed from the stomach by blotting on tissue. The empty stomachs were then weighed. The amount of the test meal that had remained in the stomach was then calculated by subtraction of successive weighings. The percentage of 3 g test meal remaining in the stomach after 30 min was calculated. Copraphagic animals or rats with food particles remaining in the stomachs were excluded from the results. Data were analysed using one-way ANOVA for independent samples followed by Tukey–Kramer test. Results and Discussion Devazepide (0.03–3.0 mg/kg) had no effect on gastric emptying (F (5,41) Å 0.6, NS) (Table 1). As Fig. 1 shows, dl -fenfluramine (3 mg/kg) significantly reduced gastric emptying (F (4,22) Å 9.7, p õ 0.0001) with 62.3% of the test meal remaining in the stomach compared with 31.5% in the control condition. Devazepide (0.1 mg/kg) substantially attenuated the effect of dl-fenfluramine. At the higher dose of 0.3 mg/kg devazepide completely reversed the effect of dl -fenfluramine (p õ 0.05), restoring gastric emptying to that of the vehicle-treated group. The results showed that devazepide blocked d l -fenfluramine-induced suppression of gastric emptying in 17 h fooddeprived rats. Under these conditions and over this range of doses devazepide has no intrinsic effect on gastric emptying. This finding suggests that d l -fenfluramine-induced suppression of gastric emptying may, in part at least, be mediated by the action of endogenous CCK acting at CCK-A receptors. TABLE 1 THE LACK OF EFFECT OF DEVAZEPIDE ON GASTRIC EMPTYING OF A SEMI-SOLID MEAL IN 17 h FOOD DEPRIVED RATS Dose Devazepide (mg/kg)
0 0.03 0.1 0.3 1.0 3.0
Percentage Test Meal Remaining in the Stomach
25.8 { 25.2 { 18.0 { 18.0 { 20.0 { 20.4 {
3.9 3.8 3.8 3.9 4.6 3.5
Data are mean { SEM. Devazepide or vehicle was administered SC 30 min prior to receiving the test meal. n ¢ 6 per group.
/ eh11 5015 Mp 546 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
CCK-5-HT INTERACTIONS
547
FIG. 1. Effect of devazepide (0.03–0.3 mg/kg) on the suppression of gastric emptying by dl-fenfluraminefenfluramine (3.0 mg/ kg) in 17 h food-deprived rats. n ¢ 5 per condition. Rats were injected with devazepide or its vehicle and 10 min later with vehicle or dl-fenfluramine 20 min before receiving the test meal. Results are shown as percentage of test meal remaining in the stomach / SEM after 30 min. Levels of significance for comparisons with vehicle condition: *p õ 0.05; ** p õ 0.01 (Tukey–Kramer). Significant difference between dl-fenfluramine group and dl-fenfluramine plus devazepide group: / p õ 0.05; // p õ 0.01 (Tukey– Kramer).
Possibly, therefore, an increase in serotonin release produced by d l -fenfluramine may bring about increased release or activity of endogenous CCK, which leads in turn to a reduction in gastric emptying. However, there is no evidence available at present to indicate that d l -fenfluramine evokes release of endogenous CCK. The method of measuring gastric emptying in the present study is based on a technique developed by Droppleman and colleagues (7). The method uses a measure of gastric retention to estimate gastric emptying. This method does not eliminate the possibility that gastric secretions may contribute to the measured gastric contents, nevertheless this technique has been shown to be reproducible and highly reliable in comparison with other tests and to be sensitive to both increases and decreases in gastric emptying (7,12). The mechanism through which CCK and serotonin may interact in the control of gastric emptying has yet to be defined . It has been suggested that CCK-8 produces a decrease in the rate of gastric emptying by causing contraction of the pyloric sphincter and devazepide is known to reverse this effect ( 16,18 ) . A recent study, which indicates that 5-HT produced contractions of the pyloric sphincter, provides evidence which argues against the two systems interacting through this mechanism ( 9 ) . The work of Eberle-Wang and Simansky suggests that 5-HT and CCK increase pyloric contractions by pharmacologically distinct mechanisms ( 9 ) . Conceivably, therefore, their results suggest that the observations in the present
study may not be due to an interaction between the two systems but reflect independent effects. Nevertheless, this appears unlikely since devazepide, when administered alone, had no intrinsic effect on gastric emptying. Resolution of these apparently conflicting results may be that the mechanism by which dl-fenfluramine suppresses gastric emptying, unlike 5-HT, may not be independent of CCK mediation. In support of this, previously it was observed that dl-fenfluramine and 5-HT produced different effects on gastric emptying (12). It was concluded that dl-fenfluramine and 5-HT may act through different mechanisms. Other authors have also suggested that the effects of 5-HT and dl -fenfluramine may be mediated by separate mechanisms (11). The anorectic effects of both CCK and 5-HT are proposed, at least in part, to be due to their inhibitory effect on gastric emptying. The present study provides strong evidence for an interaction between serotonin and CCK in the control of gastric emptying. Thus we may expect that a CCK-5-HT interaction in the control of gastric emptying may carry through to a corresponding effect on ingestive behaviour. Therefore, Experiment 2 was designed to investigate possible CCK-5-HT interactions in the control of food intake. EXPERIMENT TWO Effect of devazepide on dl -fenfluramine-induced suppression of food intake in 17 h food deprived rats.
/ eh11 5015 Mp 547 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
548
FRANCIS, DOURISH AND COOPER
FIG. 2. The lack of effect of devazepide (0.03–3.0 mg/kg) on the suppression of food intake by dl -fenfluramine (3.0 mg/kg) in 17 h food-deprived rats. n ¢ 6 per condition. Rats were injected with devazepide or its vehicle and 30 min later with vehicle or dl-fenfluramine 20 min before feeding test. Results are shown as mean intake (g) / SEM after 30 min. Levels of significance for comparisons with vehicle condition: *p õ 0.05; ** p õ 0.01 (Tukey– Kramer). Significant difference between dl-fenfluramine group and dl-fenfluramine plus devazepide group: / p õ 0.05; // p õ 0.01 (Tukey–Kramer). METHOD AND MATERIALS
Animals Twenty-seven adult male Sprague–Dawley rats (Charles River), weighing between 250–300g at the time of testing, were housed under conditions described previously and tested in their home cages. Prior to testing they were food-deprived overnight (approx. 17 h) with ad lib access to water. Drugs The doses of drugs used in this experiment were: dl-fenfluramine (3 mg/kg) or vehicle (saline), devazepide (0.03, 0.1, 0.3, 1.0 and 3.0 mg/kg) or vehicle (carboxymethylcellulose). Devazepide was administered SC 30 min before testing, while dl fenfluramine was administered IP 20 min before the rats were allowed access to the food. Procedure Following the administration of drugs, the rats were allowed access to a weighed amount of their standard diet (Heygate 41B, Heygate and Sons). The food pellets were composed of 2.5% fat, 16.5% protein and 38% carbohydrate, the remainder was nonnutrient bulk. The diet was briefly removed, reweighed and replaced 30 min and 60 min after presentation. Any spillage was collected and appropriate corrections made to weighings. Food intake was measured to the nearest 0.1 g and calculated by subtraction of successive weighings. Data were analysed using oneway ANOVA for independent samples followed by the Tukey– Kramer test.
Results and Discussion dl-fenfluramine (3 mg/kg) significantly reduced food intake in 17 h food-deprived rats (F (6,43) Å 4.3,p õ 0.002). Fig. 2 confirms that dl-fenfluramine (3 mg/kg) reduced consumption of the diet to approximately 25% of the control condition. The Tukey–Kramer test revealed that devazepide (0.03–3.0 mg/kg) failed to attenuate the anorectic effect of dl -fenfluramine. The dose of dl-fenfluramine used in the present studies was chosen for two reasons. Firstly, in our hands, this dose (3 mg/ kg) had previously been shown to produce a reliable and significant suppression of both gastric emptying and food intake (12). Using a similar dose in both experiments of the present study would allow a more direct comparison to be drawn between changes in gastric emptying and food intake. Secondly, studies in our laboratories have shown that devazepide will attenuate the anorectic effect of the same dose of the more potent d-isomer of fenfluramine (4). The present work showed that devazepide had no effect on dl-fenfluramine-induced suppression of food intake in 17 h fooddeprived rats. It is unlikely that the lack of effect of devazepide was due to an insufficient dose of antagonist, since a similar range of doses of devazepide was previously reported to reverse CCKinduced suppression of feeding (6). This finding suggests that there is no interaction between serotonin and CCK in the control of food intake. This result stands in contrast to that of Cooper and colleagues (1990) which indicated that devazepide (0.03 and 0.1 mg/kg) attenuated the anorectic effect of d-fenfluramine (3 mg/kg) (4). It is possible that procedural differences may account for the
/ eh11 5015 Mp 548 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
CCK-5-HT INTERACTIONS contrasting results. In the present study the racemic form of fenfluramine was used and not the d-isomer. The d-isomer has been shown to be more potent than the l -isomer in reducing food intake in rats (13). Since, in Cooper and colleagues study devazepide attenuated the anorectic effect of d-fenfluramine (3 mg/ kg) it may be expected that devazepide would block the anorectic effect of the same dose of the racemic compound, which is composed in part of the less potent l-isomer. A possible explanation of these apparently conflicting results is that dl -fenfluramine, unlike d-fenfluramine, may reduce food intake by a mechanism which is independent of CCK mediation. However, there are a number of other procedural differences between the studies which could also account for the contrasting results. In their experiments, non-deprived rats that had been trained to consume a highly palatable sweet mash diet were used so that a high baseline level of food intake was obtained. It is clear however that devazepide does not reverse the anorectic effect of fenfluramine under all conditions. Subsequently Cooper and Dourish (1990) reported that devazepide could also attenuate 5-HT-induced suppression of food intake under similar conditions, providing further support for a 5-HT/CCK interaction (5). However, results consistent with those of the present study but in contrast to those of Cooper and colleagues (1990) have been reported by other authors (10,15). Eberle-Wang and Simansky (1992) found no effect of devazepide on 5-HT-induced suppression of feeding in 3 h food-deprived rats consuming a sweet mash meal (10). These are similar conditions to those employed by Cooper and colleagues (1990) and at present the reasons for those contrasting results and unclear. In addition, Li and Rowland (1994) reported that, under conditions similar to the present study, devazepide did not attenuate d-fenfluranmine-induced suppression of feeding in 22 h fooddeprived rats (15). There is evidence from studies using other feeding paradigms which support 5-HT/CCK interactions. Clifton and Cooper (1992) demonstrated that devazepide attenuated the reduction in meal size produced by d-fenfluramine (3). However, devazepide did not attenuate a reduction in the frequency of feeding bouts produced by d-fenfluramine, and as a consequence the anorectic effect of d-fenfluramine persisted. Their evidence suggests that when considering interactions between 5HT/CCK it may not be appropriate to measure total food intake in some instances since more subtle changes in feeding behaviour may be occurring. GENERAL DISCUSSION
An important result to emerge from this work is that the CCKA receptor antagonist devazepide blocked the suppression of gastric emptying which dl-fenfluramine produced in 17 h food-deprived rats. This finding strongly suggests that CCK may be secondarily involved in dl-fenfluramine’s action which acts initially via serotonergic mechanisms. Since CCK, itself, is known to delay gastric emptying (17), it appears probable that dl -fenfluramine may produce effects which lead to increased endogenous CCK activity and thus impede gastric emptying. Previously, Baker and her colleagues (1) had shown that dfenfluramine blocked an increase in gastric emptying produced by devazepide. In their study, it should be noted that both drugs were administered in doses that had intrinsic effects on the rate of gastric emptying. Thus, devazepide significantly increased, while d-fenfluramine significantly decreased the rate of gastric emptying. These authors observed that antagonism of devazepide’s effects by d-fenfluramine was more than a simple addition of the two drugs’ separate effects. D-fenfluramine produced a full inhibition of gastric emptying as well as reversing the effect
549 of devazepide. This implies that devazepide should not be able to antagonise the effects of dl-fenfluramine, as we have shown presently. However, it should be recognised that there are several differences between the study of Baker et al. and the present one. In their study, the rats were only 4 h food-deprived; the test meal was a solid pellet dish; gastric emptying was measured after 2 h; d-fenfluramine was used instead of the racemic form; devazepide itself produced an increase in gastric emptying. Clearly, it is the case that devazepide does not under all conditions, block fenfluramine’s retarding effect on gastric emptying. Nevertheless, the positive evidence which we have now produced does indicate that, under appropriate conditions, fenfluramine’s effect depends upon a coupling with CCK mechanisms. We can propose, therefore, that there is not an obligatory coupling between the two, but that, under some circumstances, fenfluramine can interact with endogenous CCK. It has now been suggested that CCK-8 produces a decrease in the rate of gastric emptying by causing contraction of the pyloric sphincter, an effect which can be antagonised by devazepide (16,18). Since 5-HT may affect contraction of the pyloric sphincter (9), a possible substrate for interactions between CCK and 5-HT affecting gastric emptying rate may involve this sphincter. Nevertheless, work of EberleWang and Simansky (9) suggests that 5-HT and CCK may increase pyloric contractions by pharmacologically-distinct mechanisms. However, one should exercise some caution in interpretation, if, as we suggest, coupling between 5-HT and CCK mechanisms is not obligatory. Conditions pertaining within a study may determine whether or not the interaction between 5HT and CCK mechanisms is functional. The next important question to consider is the relationship between gastric emptying and the control of food intake. We began the introduction by referring to earlier proposals that the anorectic effects of both CCK and dl-fenfluramine may depend, in part at least, on their respective suppressant effects on gastric emptying (16,21). Later reports of interaction between CCK and 5-HT mechanisms in the control of food ingestion (3,4,5,14,20,21), therefore, may be reduced to interactions occurring at the level of the control of gastric emptying. Since the first experiment, which we report here, gave evidence for just such an interaction, one might be optimistic that an explanation couched solely in terms of effects on gastric emptying would be sufficient to account for effects on food ingestion. Nevertheless, the results of the second experiment argue against a single type of CCK-5-HT interaction sufficient to account for feeding data and the results of gastric emptying. We found that devazepide had no effect on dl -fenfluramine-induced suppression of food intake in 17 h food-deprived rats. It is unlikely that devazepide’s lack of effect was due to an insufficient dose of the antagonist, since a similar range of doses of devazepide has been reported to reverse CCK-induced suppression of feeding (6). We have to conclude that, under the conditions of Experiment 2 that any interaction occurring between 5-HT and CCK in the control of gastric emptying may not necessarily feed through to an interaction appearing in the control of food intake. It may be simpler to envisage that interactions between CCK and 5-HT may arise, independently, to affect the control of food ingestion on the one hand and gastric emptying on the other. In conclusion, our data provide evidence for an interaction between 5-HT and CCK mechanisms at the level of the control of gastric emptying. The effect of dl -fenfluramine to reduce the rate of emptying was blocked by devazepide, a selective CCKA receptor antagonist. We did not find evidence for a similar interaction affecting food ingestion in 17 h food-deprived rats. Hence, the interaction influencing gastric emptying appears not to carry through to the control of ingestive behaviour.
/ eh11 5015 Mp 549 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
550
FRANCIS, DOURISH AND COOPER REFERENCES
1. Baker, B.; Cooper, S. J.; Booth, D. A. Effects of the CCK receptor antagonist MK 329 and ( / )-fenfluramine on gastric emptying in the rat. Br. J. Pharmacol. 948:937P; 1989. 2. Boden, P. R.; Woodruff, G. N.; Pinnock, R. D. Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurons in the dorsal raphe of the rat brain. Br. J. Pharmacol. 102:635–638; 1991. 3. Clifton, P. G.; Cooper, S. J. CCK-5-HT interactions influence meal size in the free feeding rat. In: Dourish, C. T.; Cooper, S. J.; Iversen, S. D; Iversen, L. L.,eds. Multiple Cholecystokinin Receptors in the CNS; Oxford University Press; 1992:287–289 4. Cooper, S. J.; Dourish, C. T.; Barber, D. J. Reversal of the anorectic effect of ( / )-fenfluramine in the rat by the selective cholecystokinin receptor antagonist MK 329. Br. J. Pharmacol. 99:65–70; 1990. 5. Cooper, S. J.; Dourish, C. T. Multiple cholecystokinin (CCK) receptors and CCK-monoamine interactions are instrumental in the control of feeding. Physiol. Behav. 48:849–857; 1990. 6. Dourish, C. T.; Coughlan, J.; Hawley, D.; Clarke, M.; and Iversen, S. D. Blockade of CCK-induced hypophagia and prevention of morphine tolerence by the CCK antagonist L-364,718. In: Wang, R. Y.; Schoenfeld, R., eds. Cholecystokinin Antagonists, New York: Alan R. Liss; 1988:307–325. 7. Droppleman, D. A.; Gregory. R. L.; Reevis, S. A. A simplified method for assessing drug effects on gastric emptying in rats. J. Pharmacol. Meth. 4:227–230; 1980. 8. Ebenezer, I. S.; Brooman, J. Pretreatment with the 5-HT 1A receptor agonists 8-OH-DPAT or gepirone does not attenuate the inhibitory effect of systemically administered cholecystokinin (CCK) on food intake in rats. Meth. Find. Exp. Clin. Pharmacol. 16:589–595; 1994. 9. Eberle-Wang, K.; Braun, B. T.; Simansky, K. J. Serotonin contracts the isoloated rat pylorus via a 5-HT 2-like receptor. Am. J. Physiol. 266:R284–R291; 1994. 10. Eberle-Wang, K.; Simansky, K. J. The CCK-A receptor antagonist devazepide, blocks the anorectic action of CCK but not peripheral serotonin in rats. Pharmacol. Biochem. Behav. 43:943–947; 1992. 11. Fletcher, P. J.; Burton, M. J. The anorectic action of peripherally administered 5-HT is enhanced by vagotomy. Physiol. Behav. 34:943–947; 1985
12. Francis, J.; Critchley, D. J. P.; Dourish, C. T.; Cooper, S. J. Comparison between the effects of 5-HT and dl-fenfluramine on food intake and gastric emptying in the rat. Pharmacol. Biochem. Behav. 50:581–585; 1995. 13. Garattini, S., Caccia, S., Mennini, T., Samanin, R., Consolo, S., Ladinsky, H. Biochemical pharmacology of the anorectic drug fenfluramine: A review. Curr. Med. Res. Opin. 6(Suppl. 1):15–27; 1979. 14. Grignaschi, G.; Mantelli, B.; Fracasso, C.; Anelli, M.; Caccia, S.; Samanin, R. Reciprocal interaction of 5-hydroxytryptamine and cholecystokinin in the control of feeding patterns in rats. Br. J. Pharmacol. 109:491–494; 1993. 15. Li, B. H.; Rowland, N. E. Cholecystokinin and dexfenfluramineinduced anorexia compared using devazepide and c-fos expression in the rat brain. Regulatory Peptides 50:223–333; 1994. 16. Margolis, R. L.; Moran, T. H.; McHugh, P. R. In vitro response of rat gastrointestinal segments to cholecystokinin and bombesin. Peptides 10:157–161; 1989. 17. Moran, T. H.; McHugh, P. R. Gastric and non-gastric mechanisms for the satiety action of cholecystokinin. Am. J. Physiol. 254:R628– R632; 1988. 18. Murphy, R. B.; Smith, G. P.; Gibbs, J. Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat pylorus. Peptides 8:27–134; 1987. 19. Neill, J. C.; Cooper, S. J. Evidence that d-fenfluramine anorexia is mediated by 5-HT 1 receptors. Psychopharmacology 97:213–218; 1989. 20. Poeschla, B.; Gibbs, J.; Simansky, K. J.; Greenberg, D.; Smith, G. P. Cholecystokinin-induced satiety depends on the activation of the 5-HT 1C receptors. Am. J. Physiol. 264:R62–R64; 1993. 21. Poeschla, B.; Gibbs, J.; Simansky, K. J.; Smith, G. P. The 5-HT 1A agonist 8-OH-DPAT attenuates the satiating action of cholecystokinin. Pharmacol. Biochem. Behav. 42:541–543; 1992. 22. Rowland, N.; Carlton, J. Inhibition of gastric emptying by peripheral and central fenfluramine in rats: Correlation with anorexia. Life Sci. 34:2495–2499; 1984. 23. Stallone, D.; Nicolaidis, S.; Gibbs, J. Cholecystokinin-induced anorexia depends on serotonergic function. Am. J. Physiol. 256:R1138–R1141; 1989.
/ eh11 5015 Mp 550 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
PII S0031-9384(97)00015-2
Devazepide Attenuates dl-Fenfluramine-Induced Suppression of Gastric Emptying but Not Food Intake in the 17 h Food-Deprived Rat JOHN FRANCIS,* 1 COLIN T. DOURISH† AND STEVEN J. COOPER‡ *Centre for Human Nutrition, University of Sheffield, Northern General Hospital, Sheffield, S5 7AU, UK, †Cerebrus, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7PN, UK, and ‡Department of Psychology, University of Durham, Science Laboratories, South Road, Durham, DH1 3LE, UK Received 19 June 1995; Accepted 29 January 1997 FRANCIS, J., C. T. DOURISH, AND S. J. COOPER, Devazepide attenuates dl- fenfluramine-induced suppression of gastric emptying but not food intake in the 17 h food-deprived rat. PHYSIOL BEHAV 62(3) 545–550, 1997.—Recently a number of studies have provided evidence which suggests that CCK and 5-HT interact in the control of food intake. The present experiments further examine this mechanism and the possibility that CCK and 5-HT interact in the control of gastric emptying. The selective CCK-A receptor antagonist, devazepide, (0.03–3.0 mg/kg) administered alone had no intrinsic effect on gastric emptying. Devazepide (0.1 and 0.3 mg/kg) blocked dl-fenfluramine-induced (3.0 mg/kg) suppression of gastric emptying. However, devazepide (0.03–3.0 mg/kg) failed to attenuate the anorectic effect of the same dose of dl-fenfluramine. These results suggest that under the present experimental conditions CCK and 5-HT interact in the regulation of gastric emptying but not food intake. Thus the interaction between CCK and 5-HT in the regulation of gastric emptying appears not to affect the control of ingestive behaviour. q 1997 Elsevier Science Inc.
vazepide, reversed the anorectic effect of d -fenfluramine ( 4 ) . These data suggested that serotonergic influences on control of feeding may depend upon the mediation of CCK. Later, Cooper and Dourish also reported that devazepide attenuated the anorectic effect of exogenously administered 5-HT ( 5 ) . Clifton and Cooper extended this work and, using a meal pattern analysis, demonstrated that in freely-feeding rats devazepide antagonised the reduction in meal size produced by d fenfluramine ( 3 ) . This result was confirmed in later work by Grignaschi and colleagues ( 14 ) . Hence, the effect of d-fenfluramine to reduce meal size appears to be due, in part, to an action of CCK at CCK-A receptors. Evidence from electrophysiological studies also suggest that there are potential interactions between CCK and serotonin. Thus, Boden and colleagues recorded intracellularly from neurones in the dorsal raphe nucleus (2). Some of the neurones which were inhibited by 5-HT (a characteristic of serotonergic neurones) were excited by CCK-8 but not by the selective CCKB agonist, pentagastrin. Moreover, the excitatory effect of CCK8 was blocked by devazepide but not by the selective CCK-B receptor antagonist, L-365,260. Hence, CCK may act at CCK-A receptors to influence activity of 5-HT-containing neurones in the dorsal raphe. The anorectic effect of both cholecystokinin (CCK) and dl fenfluramine, an indirectly-acting serotonin (5-HT) agonist, may
RECENT studies have identified reciprocal interactions between cholecystokinin (CCK) and serotonin (5-HT) in their effects on food intake. Thus, Stallone and colleagues were the first to show that metergoline, a 5-HT receptor antagonist, attenuated the anorectic effect of CCK-8 in the 3 h food-deprived rat (23). Metergoline also reversed the anorectic effect of dl -fenfluramine, an effect which is believed to be mediated by central 5-HT 1 receptors (19). Furthermore, Stallone and colleagues showed that the anorectic effect of CCK-8 was not affected by the peripheral 5HT receptor antagonist, xylamidine. They proposed that exogenous CCK-8 acts peripherally, presumably to produce a vagallymediated signal, to alter central 5-HT activity and hence induce a satiety effect (23). Subsequently, Poeschla and colleagues and Grignaschi and colleagues confirmed the results reported by Stallone and colleagues (14,20,21). Inhibiting central 5-HT activity, through, for example, the action of selective 5-HT 1A agonist, would be expected to oppose the anorectic effect of exogenous CCK. Poeschla and colleagues reported that 8-OH-DPAT, 5HT 1A agonist, did attenuate the feeding-suppressant effect of exogenous CCK-8 (21). However, it should be noted that Ebenezer and Brooman failed to replicate this result (8). In contrast to the mediation of CCK-8-induced satiety via 5-HT mechanisms, there are data which indicate an interaction in the opposite direction. Thus, Cooper and colleagues demonstrated that the selective CCK-A receptor antagonist, de1
To whom requests for reprints should be addressed.
545 / eh11 5015 Mp 545 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
546
FRANCIS, DOURISH AND COOPER
depend, at least in part, on their inhibitory effect on gastric emptying (17,22). This apparent overlap in the effects of CCK and 5-HT on food intake and gastric emptying suggests that interactions may occur between these two transmitters to produce, in effect, a common mechanism underlying their effects. The aim of the first experiment, reported here, was to examine the possible effect of devazepide on the suppression of gastric emptying produced by dl -fenfluramine (10,21). If there is a mechanism in common, and there is some coupling between CCK- and 5-HT-dependent processes, respectively, then CCK receptor blockade may impede dl -fenfluramine’s effect. In addition, it was first necessary to establish under the present conditions the effects of devazepide administered alone on gastric emptying. The second study investigated whether devazepide would attenuate or block dl -fenfluramine-induced anorexia in food-deprived rats. EXPERIMENT ONE The effect of devazepide on dl-fenfluramine-induced suppression of gastric emptying in 17 h food-deprived rats. METHOD AND MATERIALS
Animals Seventy-four adult male Sprague–Dawley rats (Charles River, Manston, UK), weighing between 250–300 g at the time of testing, were used. They were housed individually with ad libitum access to food pellets (Diet 41B, Heygate and Sons, Northants, U.K.). They were maintained under a 12 h L:D cycle with lights on at 0800 h. Prior to testing animals were transferred to test cages, where they were food-deprived overnight (approx. 17 h) with ad lib access to water. Drugs dl-Fenfluramine was purchased from Sigma Chemical Co. Ltd. (Poole, UK). dl-fenfluramine was dissolved in isotonic saline. Devazepide was kindly donated by Merck, Sharp and Dohme (Harlow, UK). Devazepide was suspended in 0.5% carboxymethylcellulose. Drugs were administered in a volume of 1ml/kg (doses refer to base). Test Meal Carboxymethylcellulose (low viscosity, 10–20 cps, Sigma Chemical Co. Ltd., Poole, UK) was added in four 5-g portions (20 g in total) to 250 ml of distilled water. After the addition of each portion, the mixture was agitated in a blender for 1 min. Casein (bovine milk: Sigma Chemical Co. Ltd.) was added to this mixture in two 8-g portions (16 g in total), followed by 8 g of cane sugar (household; Tate and Lyle, London, UK) and 8 g cornstarch (Sigma Chemical Co. Ltd.). Following the addition of each portion of these ingredients, the mixture was agitated for 1 min. The resulting mixture was a white semisolid paste, which was placed in a refrigerator overnight to allow any air trapped in the mixture to escape. Two hours before use, the mixture was removed from the refrigerator and allowed to reach room temperature. Procedure To investigate the intrinsic effect of devazepide on gastric emptying, groups of rats ( n ¢ 6 per group ) were injected subcutaneously ( SC ) with devazepide ( 0.03, 0.1, 0.3, 1.0 and 3.0 mg / kg ) or vehicle ( carboxymethylcellulose ) 30 min prior to testing.
Previous experiments in our laboratories have shown that 3 mg/kg dl -fenfluramine significantly and reliably suppresses gastric emptying and food intake in 17h food-deprived rats, and therefore, this dose was used in the present studies (12). The doses of devazepide used had no intrinsic effect on gastric emptying and were chosen on the basis of the results of the first experiment. Thus, groups of rats, (n ¢ 5 per group) were injected (SC) 30 min before testing with devazepide (0.03, 0.1 and 0.3 mg/kg) or vehicle, while dl-fenfluramine (3 mg/kg) or vehicle (saline) were injected intraperitoneally 20 min before receiving the test meal. Following administration of drugs rats were given by gavage a weighed amount of test meal (approx. 3 g) equalling 3 ml in volume. Thirty min later, the rats were killed by cervical dislocation and laparotomized. The stomachs were ligated at the oesophagus and pylorus, to prevent leakage, removed and weighed. The stomachs were then opened and any remaining test meal was rinsed out. Any excess water was removed from the stomach by blotting on tissue. The empty stomachs were then weighed. The amount of the test meal that had remained in the stomach was then calculated by subtraction of successive weighings. The percentage of 3 g test meal remaining in the stomach after 30 min was calculated. Copraphagic animals or rats with food particles remaining in the stomachs were excluded from the results. Data were analysed using one-way ANOVA for independent samples followed by Tukey–Kramer test. Results and Discussion Devazepide (0.03–3.0 mg/kg) had no effect on gastric emptying (F (5,41) Å 0.6, NS) (Table 1). As Fig. 1 shows, dl -fenfluramine (3 mg/kg) significantly reduced gastric emptying (F (4,22) Å 9.7, p õ 0.0001) with 62.3% of the test meal remaining in the stomach compared with 31.5% in the control condition. Devazepide (0.1 mg/kg) substantially attenuated the effect of dl-fenfluramine. At the higher dose of 0.3 mg/kg devazepide completely reversed the effect of dl -fenfluramine (p õ 0.05), restoring gastric emptying to that of the vehicle-treated group. The results showed that devazepide blocked d l -fenfluramine-induced suppression of gastric emptying in 17 h fooddeprived rats. Under these conditions and over this range of doses devazepide has no intrinsic effect on gastric emptying. This finding suggests that d l -fenfluramine-induced suppression of gastric emptying may, in part at least, be mediated by the action of endogenous CCK acting at CCK-A receptors. TABLE 1 THE LACK OF EFFECT OF DEVAZEPIDE ON GASTRIC EMPTYING OF A SEMI-SOLID MEAL IN 17 h FOOD DEPRIVED RATS Dose Devazepide (mg/kg)
0 0.03 0.1 0.3 1.0 3.0
Percentage Test Meal Remaining in the Stomach
25.8 { 25.2 { 18.0 { 18.0 { 20.0 { 20.4 {
3.9 3.8 3.8 3.9 4.6 3.5
Data are mean { SEM. Devazepide or vehicle was administered SC 30 min prior to receiving the test meal. n ¢ 6 per group.
/ eh11 5015 Mp 546 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
CCK-5-HT INTERACTIONS
547
FIG. 1. Effect of devazepide (0.03–0.3 mg/kg) on the suppression of gastric emptying by dl-fenfluraminefenfluramine (3.0 mg/ kg) in 17 h food-deprived rats. n ¢ 5 per condition. Rats were injected with devazepide or its vehicle and 10 min later with vehicle or dl-fenfluramine 20 min before receiving the test meal. Results are shown as percentage of test meal remaining in the stomach / SEM after 30 min. Levels of significance for comparisons with vehicle condition: *p õ 0.05; ** p õ 0.01 (Tukey–Kramer). Significant difference between dl-fenfluramine group and dl-fenfluramine plus devazepide group: / p õ 0.05; // p õ 0.01 (Tukey– Kramer).
Possibly, therefore, an increase in serotonin release produced by d l -fenfluramine may bring about increased release or activity of endogenous CCK, which leads in turn to a reduction in gastric emptying. However, there is no evidence available at present to indicate that d l -fenfluramine evokes release of endogenous CCK. The method of measuring gastric emptying in the present study is based on a technique developed by Droppleman and colleagues (7). The method uses a measure of gastric retention to estimate gastric emptying. This method does not eliminate the possibility that gastric secretions may contribute to the measured gastric contents, nevertheless this technique has been shown to be reproducible and highly reliable in comparison with other tests and to be sensitive to both increases and decreases in gastric emptying (7,12). The mechanism through which CCK and serotonin may interact in the control of gastric emptying has yet to be defined . It has been suggested that CCK-8 produces a decrease in the rate of gastric emptying by causing contraction of the pyloric sphincter and devazepide is known to reverse this effect ( 16,18 ) . A recent study, which indicates that 5-HT produced contractions of the pyloric sphincter, provides evidence which argues against the two systems interacting through this mechanism ( 9 ) . The work of Eberle-Wang and Simansky suggests that 5-HT and CCK increase pyloric contractions by pharmacologically distinct mechanisms ( 9 ) . Conceivably, therefore, their results suggest that the observations in the present
study may not be due to an interaction between the two systems but reflect independent effects. Nevertheless, this appears unlikely since devazepide, when administered alone, had no intrinsic effect on gastric emptying. Resolution of these apparently conflicting results may be that the mechanism by which dl-fenfluramine suppresses gastric emptying, unlike 5-HT, may not be independent of CCK mediation. In support of this, previously it was observed that dl-fenfluramine and 5-HT produced different effects on gastric emptying (12). It was concluded that dl-fenfluramine and 5-HT may act through different mechanisms. Other authors have also suggested that the effects of 5-HT and dl -fenfluramine may be mediated by separate mechanisms (11). The anorectic effects of both CCK and 5-HT are proposed, at least in part, to be due to their inhibitory effect on gastric emptying. The present study provides strong evidence for an interaction between serotonin and CCK in the control of gastric emptying. Thus we may expect that a CCK-5-HT interaction in the control of gastric emptying may carry through to a corresponding effect on ingestive behaviour. Therefore, Experiment 2 was designed to investigate possible CCK-5-HT interactions in the control of food intake. EXPERIMENT TWO Effect of devazepide on dl -fenfluramine-induced suppression of food intake in 17 h food deprived rats.
/ eh11 5015 Mp 547 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
548
FRANCIS, DOURISH AND COOPER
FIG. 2. The lack of effect of devazepide (0.03–3.0 mg/kg) on the suppression of food intake by dl -fenfluramine (3.0 mg/kg) in 17 h food-deprived rats. n ¢ 6 per condition. Rats were injected with devazepide or its vehicle and 30 min later with vehicle or dl-fenfluramine 20 min before feeding test. Results are shown as mean intake (g) / SEM after 30 min. Levels of significance for comparisons with vehicle condition: *p õ 0.05; ** p õ 0.01 (Tukey– Kramer). Significant difference between dl-fenfluramine group and dl-fenfluramine plus devazepide group: / p õ 0.05; // p õ 0.01 (Tukey–Kramer). METHOD AND MATERIALS
Animals Twenty-seven adult male Sprague–Dawley rats (Charles River), weighing between 250–300g at the time of testing, were housed under conditions described previously and tested in their home cages. Prior to testing they were food-deprived overnight (approx. 17 h) with ad lib access to water. Drugs The doses of drugs used in this experiment were: dl-fenfluramine (3 mg/kg) or vehicle (saline), devazepide (0.03, 0.1, 0.3, 1.0 and 3.0 mg/kg) or vehicle (carboxymethylcellulose). Devazepide was administered SC 30 min before testing, while dl fenfluramine was administered IP 20 min before the rats were allowed access to the food. Procedure Following the administration of drugs, the rats were allowed access to a weighed amount of their standard diet (Heygate 41B, Heygate and Sons). The food pellets were composed of 2.5% fat, 16.5% protein and 38% carbohydrate, the remainder was nonnutrient bulk. The diet was briefly removed, reweighed and replaced 30 min and 60 min after presentation. Any spillage was collected and appropriate corrections made to weighings. Food intake was measured to the nearest 0.1 g and calculated by subtraction of successive weighings. Data were analysed using oneway ANOVA for independent samples followed by the Tukey– Kramer test.
Results and Discussion dl-fenfluramine (3 mg/kg) significantly reduced food intake in 17 h food-deprived rats (F (6,43) Å 4.3,p õ 0.002). Fig. 2 confirms that dl-fenfluramine (3 mg/kg) reduced consumption of the diet to approximately 25% of the control condition. The Tukey–Kramer test revealed that devazepide (0.03–3.0 mg/kg) failed to attenuate the anorectic effect of dl -fenfluramine. The dose of dl-fenfluramine used in the present studies was chosen for two reasons. Firstly, in our hands, this dose (3 mg/ kg) had previously been shown to produce a reliable and significant suppression of both gastric emptying and food intake (12). Using a similar dose in both experiments of the present study would allow a more direct comparison to be drawn between changes in gastric emptying and food intake. Secondly, studies in our laboratories have shown that devazepide will attenuate the anorectic effect of the same dose of the more potent d-isomer of fenfluramine (4). The present work showed that devazepide had no effect on dl-fenfluramine-induced suppression of food intake in 17 h fooddeprived rats. It is unlikely that the lack of effect of devazepide was due to an insufficient dose of antagonist, since a similar range of doses of devazepide was previously reported to reverse CCKinduced suppression of feeding (6). This finding suggests that there is no interaction between serotonin and CCK in the control of food intake. This result stands in contrast to that of Cooper and colleagues (1990) which indicated that devazepide (0.03 and 0.1 mg/kg) attenuated the anorectic effect of d-fenfluramine (3 mg/kg) (4). It is possible that procedural differences may account for the
/ eh11 5015 Mp 548 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
CCK-5-HT INTERACTIONS contrasting results. In the present study the racemic form of fenfluramine was used and not the d-isomer. The d-isomer has been shown to be more potent than the l -isomer in reducing food intake in rats (13). Since, in Cooper and colleagues study devazepide attenuated the anorectic effect of d-fenfluramine (3 mg/ kg) it may be expected that devazepide would block the anorectic effect of the same dose of the racemic compound, which is composed in part of the less potent l-isomer. A possible explanation of these apparently conflicting results is that dl -fenfluramine, unlike d-fenfluramine, may reduce food intake by a mechanism which is independent of CCK mediation. However, there are a number of other procedural differences between the studies which could also account for the contrasting results. In their experiments, non-deprived rats that had been trained to consume a highly palatable sweet mash diet were used so that a high baseline level of food intake was obtained. It is clear however that devazepide does not reverse the anorectic effect of fenfluramine under all conditions. Subsequently Cooper and Dourish (1990) reported that devazepide could also attenuate 5-HT-induced suppression of food intake under similar conditions, providing further support for a 5-HT/CCK interaction (5). However, results consistent with those of the present study but in contrast to those of Cooper and colleagues (1990) have been reported by other authors (10,15). Eberle-Wang and Simansky (1992) found no effect of devazepide on 5-HT-induced suppression of feeding in 3 h food-deprived rats consuming a sweet mash meal (10). These are similar conditions to those employed by Cooper and colleagues (1990) and at present the reasons for those contrasting results and unclear. In addition, Li and Rowland (1994) reported that, under conditions similar to the present study, devazepide did not attenuate d-fenfluranmine-induced suppression of feeding in 22 h fooddeprived rats (15). There is evidence from studies using other feeding paradigms which support 5-HT/CCK interactions. Clifton and Cooper (1992) demonstrated that devazepide attenuated the reduction in meal size produced by d-fenfluramine (3). However, devazepide did not attenuate a reduction in the frequency of feeding bouts produced by d-fenfluramine, and as a consequence the anorectic effect of d-fenfluramine persisted. Their evidence suggests that when considering interactions between 5HT/CCK it may not be appropriate to measure total food intake in some instances since more subtle changes in feeding behaviour may be occurring. GENERAL DISCUSSION
An important result to emerge from this work is that the CCKA receptor antagonist devazepide blocked the suppression of gastric emptying which dl-fenfluramine produced in 17 h food-deprived rats. This finding strongly suggests that CCK may be secondarily involved in dl-fenfluramine’s action which acts initially via serotonergic mechanisms. Since CCK, itself, is known to delay gastric emptying (17), it appears probable that dl -fenfluramine may produce effects which lead to increased endogenous CCK activity and thus impede gastric emptying. Previously, Baker and her colleagues (1) had shown that dfenfluramine blocked an increase in gastric emptying produced by devazepide. In their study, it should be noted that both drugs were administered in doses that had intrinsic effects on the rate of gastric emptying. Thus, devazepide significantly increased, while d-fenfluramine significantly decreased the rate of gastric emptying. These authors observed that antagonism of devazepide’s effects by d-fenfluramine was more than a simple addition of the two drugs’ separate effects. D-fenfluramine produced a full inhibition of gastric emptying as well as reversing the effect
549 of devazepide. This implies that devazepide should not be able to antagonise the effects of dl-fenfluramine, as we have shown presently. However, it should be recognised that there are several differences between the study of Baker et al. and the present one. In their study, the rats were only 4 h food-deprived; the test meal was a solid pellet dish; gastric emptying was measured after 2 h; d-fenfluramine was used instead of the racemic form; devazepide itself produced an increase in gastric emptying. Clearly, it is the case that devazepide does not under all conditions, block fenfluramine’s retarding effect on gastric emptying. Nevertheless, the positive evidence which we have now produced does indicate that, under appropriate conditions, fenfluramine’s effect depends upon a coupling with CCK mechanisms. We can propose, therefore, that there is not an obligatory coupling between the two, but that, under some circumstances, fenfluramine can interact with endogenous CCK. It has now been suggested that CCK-8 produces a decrease in the rate of gastric emptying by causing contraction of the pyloric sphincter, an effect which can be antagonised by devazepide (16,18). Since 5-HT may affect contraction of the pyloric sphincter (9), a possible substrate for interactions between CCK and 5-HT affecting gastric emptying rate may involve this sphincter. Nevertheless, work of EberleWang and Simansky (9) suggests that 5-HT and CCK may increase pyloric contractions by pharmacologically-distinct mechanisms. However, one should exercise some caution in interpretation, if, as we suggest, coupling between 5-HT and CCK mechanisms is not obligatory. Conditions pertaining within a study may determine whether or not the interaction between 5HT and CCK mechanisms is functional. The next important question to consider is the relationship between gastric emptying and the control of food intake. We began the introduction by referring to earlier proposals that the anorectic effects of both CCK and dl-fenfluramine may depend, in part at least, on their respective suppressant effects on gastric emptying (16,21). Later reports of interaction between CCK and 5-HT mechanisms in the control of food ingestion (3,4,5,14,20,21), therefore, may be reduced to interactions occurring at the level of the control of gastric emptying. Since the first experiment, which we report here, gave evidence for just such an interaction, one might be optimistic that an explanation couched solely in terms of effects on gastric emptying would be sufficient to account for effects on food ingestion. Nevertheless, the results of the second experiment argue against a single type of CCK-5-HT interaction sufficient to account for feeding data and the results of gastric emptying. We found that devazepide had no effect on dl -fenfluramine-induced suppression of food intake in 17 h food-deprived rats. It is unlikely that devazepide’s lack of effect was due to an insufficient dose of the antagonist, since a similar range of doses of devazepide has been reported to reverse CCK-induced suppression of feeding (6). We have to conclude that, under the conditions of Experiment 2 that any interaction occurring between 5-HT and CCK in the control of gastric emptying may not necessarily feed through to an interaction appearing in the control of food intake. It may be simpler to envisage that interactions between CCK and 5-HT may arise, independently, to affect the control of food ingestion on the one hand and gastric emptying on the other. In conclusion, our data provide evidence for an interaction between 5-HT and CCK mechanisms at the level of the control of gastric emptying. The effect of dl -fenfluramine to reduce the rate of emptying was blocked by devazepide, a selective CCKA receptor antagonist. We did not find evidence for a similar interaction affecting food ingestion in 17 h food-deprived rats. Hence, the interaction influencing gastric emptying appears not to carry through to the control of ingestive behaviour.
/ eh11 5015 Mp 549 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015
550
FRANCIS, DOURISH AND COOPER REFERENCES
1. Baker, B.; Cooper, S. J.; Booth, D. A. Effects of the CCK receptor antagonist MK 329 and ( / )-fenfluramine on gastric emptying in the rat. Br. J. Pharmacol. 948:937P; 1989. 2. Boden, P. R.; Woodruff, G. N.; Pinnock, R. D. Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurons in the dorsal raphe of the rat brain. Br. J. Pharmacol. 102:635–638; 1991. 3. Clifton, P. G.; Cooper, S. J. CCK-5-HT interactions influence meal size in the free feeding rat. In: Dourish, C. T.; Cooper, S. J.; Iversen, S. D; Iversen, L. L.,eds. Multiple Cholecystokinin Receptors in the CNS; Oxford University Press; 1992:287–289 4. Cooper, S. J.; Dourish, C. T.; Barber, D. J. Reversal of the anorectic effect of ( / )-fenfluramine in the rat by the selective cholecystokinin receptor antagonist MK 329. Br. J. Pharmacol. 99:65–70; 1990. 5. Cooper, S. J.; Dourish, C. T. Multiple cholecystokinin (CCK) receptors and CCK-monoamine interactions are instrumental in the control of feeding. Physiol. Behav. 48:849–857; 1990. 6. Dourish, C. T.; Coughlan, J.; Hawley, D.; Clarke, M.; and Iversen, S. D. Blockade of CCK-induced hypophagia and prevention of morphine tolerence by the CCK antagonist L-364,718. In: Wang, R. Y.; Schoenfeld, R., eds. Cholecystokinin Antagonists, New York: Alan R. Liss; 1988:307–325. 7. Droppleman, D. A.; Gregory. R. L.; Reevis, S. A. A simplified method for assessing drug effects on gastric emptying in rats. J. Pharmacol. Meth. 4:227–230; 1980. 8. Ebenezer, I. S.; Brooman, J. Pretreatment with the 5-HT 1A receptor agonists 8-OH-DPAT or gepirone does not attenuate the inhibitory effect of systemically administered cholecystokinin (CCK) on food intake in rats. Meth. Find. Exp. Clin. Pharmacol. 16:589–595; 1994. 9. Eberle-Wang, K.; Braun, B. T.; Simansky, K. J. Serotonin contracts the isoloated rat pylorus via a 5-HT 2-like receptor. Am. J. Physiol. 266:R284–R291; 1994. 10. Eberle-Wang, K.; Simansky, K. J. The CCK-A receptor antagonist devazepide, blocks the anorectic action of CCK but not peripheral serotonin in rats. Pharmacol. Biochem. Behav. 43:943–947; 1992. 11. Fletcher, P. J.; Burton, M. J. The anorectic action of peripherally administered 5-HT is enhanced by vagotomy. Physiol. Behav. 34:943–947; 1985
12. Francis, J.; Critchley, D. J. P.; Dourish, C. T.; Cooper, S. J. Comparison between the effects of 5-HT and dl-fenfluramine on food intake and gastric emptying in the rat. Pharmacol. Biochem. Behav. 50:581–585; 1995. 13. Garattini, S., Caccia, S., Mennini, T., Samanin, R., Consolo, S., Ladinsky, H. Biochemical pharmacology of the anorectic drug fenfluramine: A review. Curr. Med. Res. Opin. 6(Suppl. 1):15–27; 1979. 14. Grignaschi, G.; Mantelli, B.; Fracasso, C.; Anelli, M.; Caccia, S.; Samanin, R. Reciprocal interaction of 5-hydroxytryptamine and cholecystokinin in the control of feeding patterns in rats. Br. J. Pharmacol. 109:491–494; 1993. 15. Li, B. H.; Rowland, N. E. Cholecystokinin and dexfenfluramineinduced anorexia compared using devazepide and c-fos expression in the rat brain. Regulatory Peptides 50:223–333; 1994. 16. Margolis, R. L.; Moran, T. H.; McHugh, P. R. In vitro response of rat gastrointestinal segments to cholecystokinin and bombesin. Peptides 10:157–161; 1989. 17. Moran, T. H.; McHugh, P. R. Gastric and non-gastric mechanisms for the satiety action of cholecystokinin. Am. J. Physiol. 254:R628– R632; 1988. 18. Murphy, R. B.; Smith, G. P.; Gibbs, J. Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat pylorus. Peptides 8:27–134; 1987. 19. Neill, J. C.; Cooper, S. J. Evidence that d-fenfluramine anorexia is mediated by 5-HT 1 receptors. Psychopharmacology 97:213–218; 1989. 20. Poeschla, B.; Gibbs, J.; Simansky, K. J.; Greenberg, D.; Smith, G. P. Cholecystokinin-induced satiety depends on the activation of the 5-HT 1C receptors. Am. J. Physiol. 264:R62–R64; 1993. 21. Poeschla, B.; Gibbs, J.; Simansky, K. J.; Smith, G. P. The 5-HT 1A agonist 8-OH-DPAT attenuates the satiating action of cholecystokinin. Pharmacol. Biochem. Behav. 42:541–543; 1992. 22. Rowland, N.; Carlton, J. Inhibition of gastric emptying by peripheral and central fenfluramine in rats: Correlation with anorexia. Life Sci. 34:2495–2499; 1984. 23. Stallone, D.; Nicolaidis, S.; Gibbs, J. Cholecystokinin-induced anorexia depends on serotonergic function. Am. J. Physiol. 256:R1138–R1141; 1989.
/ eh11 5015 Mp 550 Monday Jul 07 10:45 PM EL–PB (v. 62, no. 3) 5015