Effects of 5-Hydroxytryptamine 3 Receptor Antagonists on Gastrointestinal Motor Activity in Conscious Dogs

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1 /91/ $03.OO/O THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 1991 by The American Society for Pharmacology and Experimental Therapeutics Vol. 256, No. 1 Printed in U.S.A. Effects of 5-Hydroxytryptamine 3 Receptor Antagonists on Gastrointestinal Motor Activity in Conscious Dogs NAOYUKI YOSHIDA, AKIYOSHI MIZUMOTO, YWI IWANAGA and ZEN ITOH GI Laboratories, College of Medical Technology, Gunma University, Maebashi, Japan Accepted for publication October 5, 1990 ABSTRACT We studied the effect of 5-hydroxytryptamine 3 (5-HT3) receptor antagonists on gastrointestinal (GI) motor activity in conscious dogs with force transducers implanted chronically. During the digestive state, GR38032F, BRL and ICS did not affect GI motor activity at all at doses up to 1 mg/kg, whereas BRL at 1.0 mg/kg iv. significantly stimulated GI motor activity from the stomach to the colon. When GR38032F, BRL or ICS was given iv. at doses of 0.1 to 1.0 mg/ kg during the phase I period, no direct effect was observed on GI motor activity, but the treatment inhibited the occurrence of the subsequent phase Ill activity in the stomach completely and inhibited it partially in the duodenum without affecting the plasma motilin concentration. GR38032F, BRL and ICS did not influence the caudal migration of phase Ill activity in the small intestine. In contrast, BRL at doses of 0.1 to HT mediates various physiologic and pharmacologic actions on GI function through the activation of four types of 5- HT receptors: 5-HT1, 5-HT2, 5-HT3 and 5-HT4 (Buchheit et at., 1985b; Dumuis et at., 1989; Richardson and Engel, 1986; Sanger, 1987a). Among them, it has been suggested that 5-HT3 receptors are widely distributed in the enteric nervous system (Hendriks et at., 1989; Pinkus et at., 1988) and may play an important role in the regulation of GI motility (Buchheit et at., 1985a,b; Fozard, 1989; Gidda et at., 1988; Gullikson et at., 1988). In fact, potent and highly selective 5-HT3 receptor antagonists ICS and GR38032F accelerate gastric emptying (Akkermans et at., 1988; Costall et at., 1987; Fozard, 1989) and inhibit cisplatin-induced emesis in animals and humans (Andrews et at., 1988; Bermudez et at., 1988; Cohen et at., 1989; Fozard, 1989; King and Sanger, 1989). However, the 5-HT3 receptor antagonists do not stimulate acetylcholine release from the cholinergic nerve terminals (Fozard, 1989; Sanger, 1987a,b; Sanger and Nelson, 1989). The effect is clearly different from that of the gastrokinetic agents metoclopramide and cisapride (Jacoby and Brodie, 1967; Schulze-Derieu, 1979; Schuurkes et at., 1985). Received for publication October 9, mg/kg iv. significantly stimulated Gl motor activity during the phase I period. On the other hand, when GR38032F, BRL or ICS was given during natural or motilin-induced phase Ill activity, the total contractions in the stomach and partial contractions in the duodenum were inhibited but the caudal propagation of phase Ill activity in the small intestine was not affected. In terms of GI motor activity, it is concluded that the compounds which have been grouped together as 5-HT3 receptor antagonists should be divided into two types: one (BRL 24924) is a Gl motor-stimulating type probably activating 5-HT4 receptors, and the other is a type which inhibits interdigestive gastric motor activity by blocking of 5-HT3 receptors existing on very specific sites involved in the regulation of the interdigestive gastric motor activity. BRL 24924, which has been classified as a 5-HT3 receptor antagonist, also accelerates gastric emptying in rats (Cooper et at., 1986; Fozard, 1989) and GI motor activity in dogs (Gullikson et at., 1988, 1989), and inhibits cisplatin-induced emesis in ferrets (Fozard, 1989; Miner et al, 1986) but, in contrast to selective 5-HT3 receptor antagonists, it stimulates acetylcholine release from the cholinergic nerve terminals (Fozard, 1989; Sanger, 1987a,b). Recently, BRL was identified as a 5- HT4 receptor agonist (Dumuis et at., 1989). At the present time, little is known about the effect of 5-HT3 receptor antagonists on GI motor activity in conscious dogs. In the present study, therefore, we used chronically implanted force transducers in conscious dogs to investigate the effects of specific 5-HT3 receptor antagonists ICS , BRL and GR38032F on GI motor activity in comparison with those of BRL 24924, which has 5-HT3 receptor antagonist and 5-HT4 agonist properties. Materials and Methods Preparation of animals. Four healthy mongrel dogs of both sexes weighing 10 to 14 kg were anesthetized with an i.v. injection of pentobarbital sodium (Nembutal, 30 mg/kg b.wt.) and the abdominal cavity was opened under aseptic conditions. Extraluminal force transducers ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; GI, gastrointestinal; IMC, interdigestive migrating contractions; lam, immunoreactive motilin. 272

2 1991 were sutured onto the serosa of the GI tract from the gastric body to the descending colon in a manner to measure circular muscle contraction, as reported previously (Itoh et at., 1977). The sites of transducer implantation were the gastric body opposite the splenic hilum, the gastric antrum 3 cm proximal to the pyloric ring, the duodenum, the jejunum, the midintestine, the terminal ileum and the proximal and distal colon. The lead wires of these transducers were taken out of the abdominal cavity and then brought out through a skin incision made between the scapulae. The outer end of the lead wires was sutured onto the skin adjacent to the skin incision. After the abdominal surgery, a 5-cm longitudinal skin incision was made in the right frontal neck to expose the external jugular vein. A Silastic tube (French size 6.5, Dow Corning, Midland, MI) was placed into the superior vena cava through the vein and sutured onto the adjacent skin as a route for the i.v. injection of test materials. The outer end of this tube was closed with a tiny plastic cap after the tube was filled with 10% heparin solution. After the operation, a jacket protector was placed on the dog to protect the lead wires and the Silastic tube. The dogs were housed in individual experimental cages and given dog food [Gaines Meal, 20 g (dry wt.)/kg b.wt., Ajinomoto-General Foods Corp., Tokyo, Japan] at 5:00 P.M. and water was given freely. Experimental procedures. GI motor activity was recorded on a polygraph (RM 6000, Nihon Kohden Kohgyo, Tokyo, Japan) by confleeting the lead wires of the transducers to the connecting cables from the amplifiers (UG , Nihon Kohden Kohgyo, Tokyo, Japan) under the protective jacket as reported previously (Itoh et at., 1977). Test materials or saline alone were given as a bolus injection through the indwelling Silastic tube during phase I and phase III in the stomach in the interdigestive state. The injection was given immediately after the first 3 maximum contractions of the phase III contractions and also given 10 mm after the termination of the phase III contractions in the stomach. We also examined the effect of test materials on the motilin-induced phase III activity. Motilin was infused i.v. by means of an infusion pump (STC-521, TERUMO, Tokyo, Japan) during the quiescent period of the interdigestive state. The infusion started 10 to 15 mm after the termination of the natural IMC in the stomach and continued for 20 mm. Test materials or saline alone were given immediately after the first 3 maximum contractions of the motilin-induced phase III. The effects of 5-HT3 receptor antagonists were also observed during the digestive state (2-3 hr after feeding) to determine whether they stimulate motor activity during that state. Measurement of plasma IRM. To measure the IRM concentration in plasma in response to test drugs, 5-HT3 receptor antagonists were administrated i.v. at 30 mm after the termination of the IMC in the Lu_ - Antrum LUL. enum GR38032F 1.0 mg/kg, 5-HI3 Receptor Antagonisti, GI Motility 273 stomach. The collection of blood samples began at the termination of the first IMC and was continued at 15-mm intervals to the termination of the next IMC and the blood was centrifuged at 3000 rpm for 10 mm after adding 0.1 ml of aprotinin (Trasylol, 10,000 kallikrein U/ml, Bayer, Tokyo, Japan). Supernatants were separated and stored at -30#{176}C until the radioimmunoassay for motilin. The IRM concentration in the plasma was measured by the specific radioimmunoassay for motilin as reported previously (Itoh et a!., 1978). Analysis of data. The same experiments were repeated at least twice in each ofthe dogs, and quantitative analysis of GI motor activity was expressed as the motor index. The motor index was represented by the area surrounded by the contraction waves and the base line during a certain fixed period; this was measured by our own system controlled by a computer (PC-9801RX, NEC Inc., Tokyo, Japan). The effect of a single bolus injection of 5-HT3 receptor antagonists on gastric motor activity in the digestive state was expressed as the motor index ratio for 30 mm between before and after the i.v. drug administration. The results were expressed as means ±S.E. Statistical analysis between control and drug-treated motor index ratios was made by means of the Williams-Wilcoxon s multirange test (MUSCOT Statistical Analysis Program, Yukms Co., Ltd., Tokyo, Japan). Drugs. The following drugs were generously donated: BRL and BRL (Beecham Pharmaceuticals, Harlow, Eseex, UK) and GR38032F (Glaxo Group Research, Hertfordshire, UK). The following drugs were purchased: synthetic motilin (Peninsula Laboratories, Inc., Belmont, CA) and ICS (Research Biochemicals, Inc., Natick, MA). Results Two weeks postoperatively, GI contractile activity could be divided into two main patterns of activity, interdigestive and digestive states. In the interdigestive state, IMC were seen to occur at regular intervals of 100 to 120 mm in the stomach and migrated through the small intestine at a constant velocity. In all animals, feeding disrupted the regular IMC pattern and initiated the digestive pattern of activity that is characterized by regularly occurring contractile activity in the stomach and irregularly occurring contractile activity in the small intestine. Effect of 5-HT3 receptor antagonists on phase I activity in the interdigestive state. When GR38032F was given i.v. at a dose of 1 mg/kg during the phase I period, no direct effect was observed on GI motor activity (fig. 1). However, the _J FIg. 1. Effect of GR38032F on GI motor activity during the interdigestive state (phase I) in a conscious dog.. -

3 274 Yoshida et al. Vol. 256 I GB D J DC 4J - GR38032F I I - ara.* a I. M.I&- AC.. Time Intervals, 30 mm Fig. 2. Effect of GR38032F on endogenous release of motilin and GI contractile activity during the interdigestive state in a conscious dog. administration of GR38032F inhibited the occurrence of the subsequent phase III activity completely in the stomach and partially in the duodenum, but did not affect propagation in the small intestine (fig. 2). In addition, GR38032F did not alter the colonic motor activity. The plasma motilin concentration during the interdigestive state was not affected by GR38032F, as shown in figure 2. Both ICS and BRL appeared to have a similar effect to GR38032F (data not shown). In contrast, BRL at doses of 0.01 to 1.0 mg/kg i.v. significantly stimulated the GI contractile activity dose relatedly from the stomach to the colon simultaneously (fig. 3). These contractile patterns induced by BRL were different from the contractile activity of naturally occurring phase zu7 BRL24924 I III contractile patterns. The motor indexes for 30 mm after drug administration were 0.11 ± 0.24 (0.01 mg/kg; P >.05), 2.76 ± 0.50 (0.1 mg/kg; P <.05) and 6.47 ± 0.30 (1.0 mg/kg; P <.01), respectively. In addition, after the BRL injection (1 mg/kg i.v.), the plasma level of motilin was increased significantly (P <.01, N = 3) from the mean basal value of ± 22.2 (pg/ml) to the peak value of ± 42.4 (pg/ml) at 10 mm. Effect of 5-HT3 receptor antagonists on phase III activity in the interdigestive state. When BRL was given i.v. at 0.02 to 0.1 mg/kg during the phase III period, the contractile activity in the gastric body and antrum was inhibited in a dose-dependent manner (figs. 4 and 6). The contractile activity in the duodenum was inhibited partially by BRL and followed immediately by phase III activity in the jejunum (fig. 4). A similar effect was observed with GR38032F and ICS (fig. 6). In contrast, BRL at a dose of 0.3 mg/kg i.v. altered the phase III activity in the stomach to weak contractile activity (data not shown). Effect of 5-HT3 receptor antagonists on the phase III activity induced by exogenous motilin. Within 10 mm after the infusion of motilin (0.3 zg/kg/hr), phase III activity was initiated in the stomach and duodenum and migrated distally. When GR38032F was given i.v. at doses of 0.01 to 0.05 mg/kg, the phase III activity induced by motilin in the stomach was inhibited in a dose-dependent manner, but the propagation of IMC in the small intestine was not disrupted (figs. 5 and 6). Similar effects of ICS and BRL were observed (fig. 6). The action of BRL (0.3 mg/kg i.v.) was similar to the naturally occurring phase III activity. Effect of 5-HT3 receptor antagonists on the digestive state. An i.v. administration of GR38032F (1.0 mg/kg i.v.) had no significant effect on GI contractile activity at 2 hr after feeding in a dog. Even when the dose of GR38032F was increased to 1.0 mg/kg i.v., no particular change was observed in GI contractile activity (fig. 7). Both ICS and BRL up to 1 mg/kg i.v. also had little effect on GI contractile activity (data not shown). In contrast, BRL 24924, 1 mg/kg, stimulated the GI contractile activity from the stomach to the colon simultaneously. The contractile response in the gastric Suenum. J&&j ijfflllith: M-Intesth,e Fig. 3. Effect of BRL on GI motor activity during the mnterdigestive state (phase I period) in a conscious dog. jill.ij Ascend Time Intervals. 10mm

4 HT3 Receptor Antagonists, GI MotIlity 275 BRL mg/kg --- Antrum 4LL LL.H4lg. li. --,. Fig. 4. Effect of BAL on the natural phase Ill activity in a conscious dog. -, Gastnc Antrum IIjJ GR38032F 0.O5mglkg [ Motilin 0.3 pg/i#{231}-ty ULkL. J _._*_..,& Jejunum Timelntervals. 10mm antrum and duodenum was remarkable, as shown in figure 8. The antral motor index for 30 mm after BRL injection (1 mg/kg i.v.) was increased to ± 10.8% (P <.05) of the preinjection value. Discussion The present study indicates that specific 5-HT3 receptor antagonists can be divided into two group, one (BRL 24924) for GI motor stimulation and the other (ICS , BRL and GR38032F), which has no GI motor-stimulating activity. BRL 24924, which has GI motor-stimulating activity, is a substituted benzamide (Cooper et at., 1986; Fozard, 1989; Sanger, 1987b). It is well known that substituted benzamides such as metoclopramide (Jacoby and Brodie, 1967; Pinder et at., 1976; Schulze-Derieu, 1979) and cisapride (Lee et al., 1984; Schuurkes et at., 1985) increase gastric emptying and GI motility in animals and man. Our experimental results suggest that BRL strongly stimulates GI motility from the stomach ----, Fig. 5. Effect of GR38032F on the motilin-induced phase III activity in a conscious dog. to the colon simultaneously. Interestingly, when BRL at 1 mg/kg i.v. was given during phase I, the plasma motilin concentration was increased significantly. The mechanism by which BRL stimulates motilin release is unclear. However, we propose that the increase in plasma motilin after i.v. administration of BRL is an epiphenomenon secondary to the contraction of the intestine directly induced by BRL 24924, because BRL induces neither motilin-like premature phase III activity nor migrating contractions in small intestine. Sanger (1987b) reported that BRL 24924, like metoclopramide, stimulates the gut motility by increasing acetylcholine release from the cholinergic nerve terminals of the gut. Our preliminary results showed that the GI motor-stimulating activity of BRL is completely abolished by atropine, an anticholinergic agent (data not shown). Recently, Dumuis et al. (1989) reported that BRL 24924, cisapride and metoclopramide were agonists at the nonclassical 5-HT receptor (called 5-HT4), which differed from the known 5-HT1, 5-HT2 and 5-HT3 receptors. The activation of 5-HT4 receptors by the benzamides can

5 276 Yoshida et al. Vol. 256 Fig. 6. Dose-dependent inhibitory effect of 5-HT3 receptor antagonists on the natural phase Ill activity (upper) and motilin-induced phase Ill activity (lower) in the stomach in conscious dogs. Values are means ± SE. for 2 trials in each of the 3 dogs. The differences from the control group (saline) that are statistically significant: 5P <.05; P <.01. :::: Jejunum Antrum Natural Phaseffi stimulate adenylate cyclase activity in mouse embryo colliculi neurons. Furthermore, it has been suggested that the adenylate cyclase-linked nonclassical 5-HT receptor is the same as the 5- HT-like receptor involved in the prokinetic effect of the benzamides. Iwanaga et at. (1989) reported that 5-HT stimulated GI motility from the stomach to the colon simultaneously during the phase I and digestive state in conscious dogs and that 5-HT-induced stimulation of GI motility was almost completely inhibited by atropine. In our preliminary findings, the GI contractile pattern induced by 5-HT was similar to that of BRL and the GI motor-stimulating activity of 5-HT and BRL were not prevented by the 5-HT3 receptor antagonist (ICS ). Therefore, it may be possible that the stimulation by BRL of GI motility is due to the activation of 5-HT4 receptors. GR38032F a A. i.. ilit. - g g #{163}. a In contrast to BRL 24924, nonbenzamide compounds such as GR38032F, BRL and ICS even at 1.0 mg/kg i.v. did not stimulate the GI motility during phase I and the digestive period. Davidson and Pilot (1986) reported that ICS did not affect GI motility in conscious fasting or fed dogs. Moreover, BRL (Sanger, 1987a; Sanger and Nelson, 1989) and ICS (Sanger, 198Th) did not facilitate cholinergic activity in isolated guinea pig or human stomach, whereas BRL potently facilitated cholinergic activity by increasing neuronal acetylcholine release (Sanger, 198Th). This evidence therefore suggests that nonbenzamide compounds are unrelated to the cholinergic mechanism. In contrast, it was suggested that ICS and GR38032F accelerated gastric emptying in guinea pigs (Buchheit et at., 1985a; Costall et at., 1987) and man (Akkermans et at., 1988). However, Gullikson et at. (1989) suggested that in the dog BRL and ICS had no effect on the gastric emptying of solids and liquids delayed by adrenergic apha-2 agonists, whereas BRL and cisapride enhanced them. Thus, these findings were in line with our data in conscious dogs. These differences may indicate that there may be a species difference between dog, guinea pig and humans or a functional difference between motility and gastric emptying. In regard to the small intestinal or colonic propulsion, Talley et at. (1988, 1989) reported that GR38032F did not alter small intestinal transit, but delayed colonic transit in humans. On the other hand, Stacher et at. (1989) reported that ICS had slight, but significant, stimulatory effects on the motor activity of the human sigmoid colon. However, in 930 did not alter the colonic motor activity during either the fed or the fasting state. It is of great interest that nonbenzamide compounds, ICS , BRL and GR38032F, strongly inhibited both the naturally occurring IMC and motilin-induced occurring IMC. The potencies of these three compounds in inhibiting the phase III activity correlated well with those of their antiemetic activity (Bermudez et at., 1988; Cohen et a.!., 1989; Fozard, 1989) and their affinities for 5-HT3 receptor bindings (Kilpatrick et at., 1989; Pinkus et at., 1988). Furthermore, these compounds selectively inhibited the phase III activity in the stomach and L i a I Fig. 7. Effect ofgr38o32f on GI motor activity during. the digestive state(2-3 hr afterfeeding) in a conscious dog. the present studies on conscious dogs GR38032F and ICS ,-.-

6 HI3 Receptor Antagonists, GI Motility 277 BRL24924 J7 GastrlcAntrum ;;; Fig. 8. Effect of BRL on GI motor activity during the digestive state (2-3 hr after feeding) in a conscious dog. LA partially in the duodenum without affecting the propagation of distally migrating contractions in the small intestine or the IMC cycle times. BRL 24924, which has GI motor-stimulating activity, did not inhibit the phase III activity and immediately caused weak contractile activity during phase III. These results suggest that the action of nonbenzamide compounds is different from that of BRL In addition, ketanserin, a specific 5- HT2 receptor antagonist, or methysergide, a 5-HT1 and 5-HT2 mixed receptor antagonist, had no effect on the phase III activity (data not shown). These results indicate that these three 5-HT3 receptor antagonists which have no stimulating GI motor activity have a specific inhibitory action on naturally occurring or motilin-induced IMC. However, it is not clear how the phase III activity in the stomach is inhibited by these three 5-HT3 receptor antagonists. One possible mechanism is thought to be the inhibition of endogenous motilin release during phase III activity. It is a well known fact that the initiation of IMC in the stomach is closely associated with the plasma concentration of motilin in the dog and humans (Itoh et at., 1977, 1978; Itoh and Sekiguchi, 1983; Lee et at., 1978; Peeters et at., 1980; Wingate et at., 1976), because exogenous motilin initiates phase III contractions in the stomach (Itoh et at., 1978; Wingate et at., 1976), and exogenous motilin antisera completely abolish the occurrence of the contractions in the stomach (Lee et at., 1983). However, these three compounds did not influence the plasma motilin concentration during the phase III activity. As another possible mechanism, these compounds may inhibit acetylcholine release from cholinergic nerves or have an antimuscarinic activity. In general, it is accepted that the phase III activity is blocked by atropine (El-Scharkawy et at., 1981; Ormsbee and Mir, 1978; Ormsbee et at., 1979). For example, atropine inhibits natural or motilin-induced phase III activity in the stomach and disrupts the propagation of distally migrating contractions in the small intestine. In addition, it is also known that anticholinergic agents strongly inhibit GI motor activity during the digestive state. In these respects, the mode of action of these 5-HT3 receptor antagonists on GI motor activity was clearly different from that of atropine. Therefore, it may be possible that these 5-HT3 receptor antagonists have the ability to interact with motilin receptor or that the effect of motilin is mediated via the 5-HT3 receptor, which is on an unidentified site in gut or other tissue. In any case, this hypothesis may provide a key in solving the mystery of the mechanism by which these three 5-HT3 receptor antagonists block the phase III activity. 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