0022-3565/97/2833-1000$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:1000 –1008, 1997
Vol. 283, No. 3
Printed in U.S.A.
Comparison of Effect of Mosapride Citrate and Existing 5-HT4
Receptor Agonists on Gastrointestinal Motility In Vivo
and In Vitro
YUKIKO MINE, TAKASHI YOSHIKAWA, SEIKO OKU, RYUJI NAGAI, NAOYUKI YOSHIDA and KANOO HOSOKI
Department of Pharmacology I, Discovery Research Laboratories I, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan
Accepted for publication July 2, 1997
The 5-HT4 receptors, first identified in fetal mouse collicular cell cultures and named by Dumuis et al. (1988), are
positively coupled to adenylyl cyclase in brain tissue (Bockaert et al., 1990, 1992) and smooth muscle (Ford et al., 1992).
5-HT4 receptor-mediated functional responses have been detected in a variety of tissues, including the guinea pig ileum
(Craig and Clarke, 1990) and colon (Elswood et al., 1991;
Wardle and Sanger, 1993), rat esophagus (Baxter et al.,
1991), sheep pulmonary vein (Cocks and Arnold, 1992) and
both pig (Villalon et al., 1990, 1991) and human heart (Kaumann et al., 1990a, b). Agonists of the 5-HT4 receptors are
currently known to include three structurally distinct chemical classes: indoles-based molecules, substituted benzamides
and benzimidazolones. Among them, substituted benzamides
such as metoclopramide, zacopride, cisapride and renzapride
and benzimidazolone derivatives such as BIMU 1 and BIMU
8 are notable agonists at 5-HT4 receptors in GI tissues (Turconi et al., 1991; Rizzi et al., 1992; Briejer et al., 1995) and
stimulate neuronally mediated cholinergic contractions in
the GI tract in vitro and in vivo (Ford and Clarke, 1993).
Mosapride citrate, a substituted benzamide, is a novel
5-HT4 receptor agonist. We have reported that mosapride
Received for publication June 25, 1996.
the receptor binding studies, mosapride inhibited [3H]GR113808 binding to 5-HT4 receptor sites of guinea pig striatum with an IC50 value of 113 nM. In addition, mosapride
caused relaxation of the carbachol-precontracted rat esophagus, enhanced the electrically evoked contractions of guinea
pig ileum and evoked the contractions of guinea pig distal colon
with EC50 values of 208, 73, and 3029 nM, respectively; this
indicates that mosapride has a low affinity for colon than for the
rest of the GI tract. In contrast, cisapride, zacopride or BIMU 8
had similar potencies in all preparations examined. In conclusion, these studies indicate that mosapride selectively stimulates upper GI motility in vivo and in vitro. These results also
suggest heterogeneity of 5-HT4 receptors in the GI tract.
increases the gastric emptying in rats, stimulates the gastric
motor activity in conscious dogs and increases the electrically
evoked contractions in isolated guinea pig ileum (Yoshida et
al., 1989, 1991). In clinical studies, mosapride alleviates such
dysfunctions in GI motility as nonulcer dyspepsia, gastroparesis, gastric stasis and gastroesophageal reflux disease (Kanaizumi et al., 1991; Yoshida et al., 1993). Its prokinetic
action derives from facilitating ACh release from neurons of
the myenteric plexus via stimulation of 5-HT4 receptors (Yoshida et al., 1991, 1993). Moreover, receptor ligand binding
studies demonstrated that mosapride showed no affinity for
dopamine D2, adrenaline a1, adrenaline a2, 5-HT1 and 5-HT2
receptors except a weak affinity for 5-HT3, whereas other
benzamides have high affinities for several of the existing
5-HT and other neurotransmitter receptors (Yoshida et al.,
1989; Karasawa et al., 1990). For example, zacopride or
BIMU 8, although they act as potent 5-HT4 receptor agonists,
also possesses high affinity for 5-HT3 receptors (Turconi et
al., 1991; Briejer et al., 1995). Cisapride is a nonselective
5-HT4 receptor agonist that possesses affinity for dopamine
D2, 5-HT2, alpha-1 adrenoceptor and muscarinic receptors
(Karasawa et al., 1990; Briejer et al., 1995). These findings
indicate that mosapride would be a more selective 5-HT4
agonist than other benzamides and benzimidazolone. On the
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; GMC, giant migrating contractions; LMMP, longitudinal muscle myenteric plexus.
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ABSTRACT
Mosapride citrate is a new gastroprokinetic agent that enhances the upper GI motility by stimulating 5-hydroxytryptamine4 (5-HT4) receptors. The purpose of this study was to
compare the effects of mosapride and the existing 5-HT4 receptor agonists on GI motility in conscious dogs and on various
5-HT4 receptor-mediated responses in vitro. In conscious dogs
with force transducers implanted, mosapride (0.3–3 mg/kg i.v.)
stimulated the antral motility without affecting the colonic motility. However, cisapride, zacopride and BIMU 8 (0.1–1 mg/kg
i.v.) stimulated both antral and colonic motility. The enhanced
GI motility induced by mosapride or cisapride was antagonized
by pretreatment with GR113808 (1 mg/kg bolus i.v., thereafter
1 mg/kg/hr infusion), a selective 5-HT4 receptor antagonist. In
1997
Mosapride Stimulates Upper GI Motility
Materials and Methods
Animals. Beagle dogs (Nihon Nohsan Kohgyo Inc., Yokohama,
Japan) of both sexes weighing 9 to 13 kg, male rats of Jcl SD strain
(Nihon Cler Inc., Osaka, Japan) weighing 180 to 300 g and guinea
pigs of the Hartley strain (Nihon SLC Inc., Shizuoka, Japan) weighing 200 to 400 g were used.
The dogs were individually housed in experimental cages and
given dog food (20 g of dry weight per kilogram of body weight,
Oriental Yeast, Tokyo, Japan) at 10:00 A.M. daily. The composition of
dog food was protein 25.7%, fat 8.6%, carbohydrates 47.3% and
minerals 4.3%. Water was available ad libitum. The rats and guinea
pigs were housed, with free access to food and water, in a room kept
at 22–24°C under a 12-hr light-dark cycle. The experiments were
carried out at a room temperature of 22–24°C.
GI motor activity in conscious dogs. Five healthy beagle dogs
of both sexes were anesthetized with pentobarbital sodium (30 mg/kg
i.v.), and the abdominal cavity was opened under sterile conditions.
Extraluminal force transducers (F-121S, Star Medical, Tokyo, Japan) were sutured onto the seromuscular layer of the gastric antrum
and ascending colon in such a way as to make it possible to measure
circular muscle contractions, as reported previously (Itoh et al.,
1977). The transducers were implanted in the gastric antrum, 3 cm
proximal to the pyloric ring, and in the ascending colon, 10 cm and 20
cm distal to the ileo-colonic junction, respectively. The lead wires of
the transducers from the abdominal cavity were brought out through
a skin incision made between the scapulae. The outer ends of the
lead wires were sutured onto the skin adjacent to the skin incision.
A Silastic tube (Fr. size 6.5, Dow Corning, Midland, MI) was placed
into the superior vena cava through the vein as a route for the i.v.
injection of test drugs, and the tube was sutured onto the adjacent
skin. After the operation, a jacket protector was placed on the dog to
protect the lead wires and the Silastic tube.
GI motor activity was recorded on a polygraph system (RTA1200M, Nihon Kohden Kohgyo, Tokyo, Japan) by connecting the lead
wires of the transducers with cables from the amplifiers (AP-100F,
Nihon Kohden Kohgyo) under the protective jacket as reported previously (Itoh et al., 1977). Two weeks after the operation, test drugs
or solvent was administered to the dogs i.v. through the implanted
Silastic tube in the postprandial state (2–3 hr after feeding). In some
experiments, GR113808, a 5-HT4 receptor antagonist (1 mg/kg bolus
i.v., thereafter 1 mg/kg/hr infusion), was administered, and then
mosapride or cisapride was i.v. administered 10 min after the start of
the infusion of GR113808.
To measure motor activity quantitatively, the motor index was
determined. The signals from the force transducers implanted in the
gastric antrum and ascending colon were analyzed by our own sys-
tem controlled by a computer (PC-9801RX, NEC, Inc., Tokyo, Japan).
The motor index given by the processing system corresponded to the
measurements of the area surrounded by the contraction wave and
base line, i.e., the product of the amplitude (voltage) and the time in
minutes over a fixed period. The motor index for the 30-min period
after the administration of test drugs is expressed as a percentage of
that for the 30-min period before administration.
Isolated rat thoracic esophageal muscularis mucosae. Rats
were killed by a blow on the head, and the most distal 2 cm of the
esophagus was removed. The esophageal segments were prepared as
described by Baxter et al. (1991). Briefly, the external muscularis
propria, containing the outer longitudinal and circular muscle layers
of the esophagus, was carefully removed in order to isolate the
smooth muscle of the tunica muscularis mucosae. The preparations
were suspended longitudinally under an initial tension of approximately 1 g in modified Krebs-Henseleit solution at 37°C and saturated with 95% O2 and 5% CO2. The ionic composition of the KrebsHenseleit solution (mM) was NaCl 118, KCl 4.75, CaCl2 2.5, KH2PO4
1.2, MgSO4 1.2, NaHCO3 25 and glucose 10. This solution routinely
contained alaproclate hydrochloride (1 mM) and corticosterone (30
mM) to prevent tissue uptake of 5-HT, methysergide (1 mM) to block
5-HT1 and 5-HT2 receptors and pargyline (100 mM) to prevent oxidation of 5-HT by monoamine oxidase. Tissues were left to equilibrate with Krebs-Henseleit solution for 60 min (with washing every
15 min) before starting the experiment. Responses were recorded
isometrically through a force displacement transducer (SB-1T, Nihon Kohden Kohgyo) coupled to a chart recorder (Servocorder SR
6221, GRAPHTEC, Tokyo, Japan).
The preparations were contracted by addition of a submaximal
concentration of carbachol (3 mM) to the bathing solution. Upon
establishment of a stable contraction, a cumulative concentrationeffect curve for relaxation to 5-HT was constructed. After the construction of the control curve, the tissues were washed with fresh
modified Krebs-Henseleit solution and allowed to recover for 60 min
before recontracting with carbachol. In agonist studies, potency relative to 5-HT was calculated from experiments in which two concentration-effect curves were constructed in the same preparation: the
first to 5-HT itself and the second to a test agonist. In antagonist
studies, a control concentration-effect curve to an agonist was constructed. The antagonist was incubated with the tissue for 45 min,
and the second concentration-effect curve was constructed in the
presence of the antagonist.
Isolated guinea pig distal colon. Guinea pigs were killed by a
blow on the head, and distal colon (approximately 7–8 cm from the
anus) was removed and placed in De Jalon’s solution of the following
composition (mM): KCl 5.6, CaCl2 0.5, NaHCO3 6.0, NaCl 155, glucose 2.8. Sections of longitudinal muscle with myenteric plexus
(LMMP; 2–3 cm in length) were dissected as previously described
(Wardle and Sanger, 1993). The preparations were suspended longitudinally under an initial tension of approximately 1 g in modified
De Jalon’s solution at 37°C saturated with 95% O2 and 5% CO2 and
containing granisetron (1 mM) and methiothepin (100 nM) to inhibit
responses mediated by 5-HT3 receptors and by 5-HT1-like and 5-HT2
receptors respectively. Responses were recorded through an isotonic
transducer (TD-112S, Nihon Kohden Kohgyo) and displayed on a
chart recorder (Servocorder SR 6221, GRAPHTEC). The preparations were allowed to stabilize for 45 min (washing every 15 min)
before the experiment was started.
The preparations were first exposed to a supramaximal concentration of carbachol hydrochloride (1 mM) to establish the maximal
responses evoked by each tissue. Then tissues were exposed to 5-HT
(1 mM) four or five times until consistent responses were obtained.
Agonist concentration-response curves were constructed noncumulatively by adding increasing concentrations of agonist at 15-min
intervals. The agonist was left in contact with the tissue until a
maximal response was obtained (usually 30 sec). In each tissue, two
or three agonist concentration-response curves were constructed. In
all experiments, the first curve was constructed to 5-HT itself, and
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other hand, the prokinetic effect of mosapride on GI motor
activity was somewhat different from that of cisapride. Specifically, mosapride selectively enhanced the motor activity of
the upper GI tract, such as of the stomach and duodenum,
whereas cisapride stimulated the motor activity in all sites of
the GI tract from the stomach to the colon in conscious dogs
(Yoshida et al., 1991). At the present time, the mechanisms
underlying the different effects of mosapride and cisapride on
lower GI motor activity remain obscure. Moreover, there are
no reports on whether the existing 5-HT4 agonists, such as
zacopride and BIMU 8, can stimulate upper and/or lower GI
motor activity in dogs. Therefore, we felt it necessary to
compare more precisely the GI motor activity of mosapride
and that of the existing 5-HT4 agonists cisapride, zacopride
and BIMU 8 in the dog. We further determined the relative
activities of these agents at 5-HT4 receptor in GI tract tissues: the rat esophagus, the guinea pig ileum and the guinea
pig colon.
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1002
Mine et al.
Drugs. The drugs used in the experiments were mosapride [(6)4-amino-5-chloro-2-ethoxy-N-(4-(4-fluorobenzyl)-2-morpholinyl)methyl)benzamide citrate], GR113808, (6)zacopride hydrochloride,
BIMU 8 hydrochloride, granisetron hydrochloride and alaproclate hydrochloride, which were synthesized at our laboratories; cisapride
(extracted from Acenalin, Kyowa Hakko Kogyo Co., Ltd., Tokyo,
Japan), 5-HT creatine sulfate (E. Merck Darmstadt, Germany); methiothepin maleate (Research Biochemical Inc., Natrick, MA); carbachol hydrochloride and pargyline hydrochloride (Sigma Chemical
Co., St. Louis, MO) and methysergide maleate (Sandoz Ltd., Basel,
Switzerland).
Mosapride and cisapride were dissolved in a solution containing
1% lactic acid or 0.1% dimethyl sulfoxide (DMSO). The other drugs
were dissolved in saline or in deionized water.
Results
In Vivo Studies
Effects of 5-HT4 receptor agonists on antral and colonic motor activity in conscious dogs. In the postprandial state, the antral motor activity was characterized by
regular contractile activity and remained constant for at
least a few hours after feeding without interdigestive migrating contractions occurring. On the other hand, the colonic
motor activity was characterized by colonic migrating or nonmigrating motor complexes and GMCs (Sarna et al., 1984;
Karaus and Sarna, 1987).
As shown in figure 1, mosapride (0.3–3 mg/kg i.v.) dosedependently increased the antral motor activity in conscious
dogs, and the maximal effect of mosapride (3 mg/kg) on
antral motor activity was equal to that of cisapride (1 mg/kg).
The antral motor activity-stimulating effect of cisapride (1
Fig. 1. Effect of i.v. administered mosapride, cisapride, zacopride and
BIMU 8 on antral motor activity in the postprandial state in conscious
dogs. The motor index for the 30-min period after i.v. administration of
test drugs is expressed as a percentage of that for the 30-min period
before administration. Each point represents the mean with the S.E.M.
in 4 to 5 dogs. *P , .05; **P , .01, significant differences from the
solvent treatment. (Student’s t test).
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the second curve was constructed to a test agonist. For antagonist
experiments, the third curve was constructed to a test agonist in the
presence of the appropriate concentration of antagonist added immediately after completion of the second curve. In all experiments, a
minimum of 45 min was left between successive curves. Responses
were expressed as a percentage of the maximal 5-HT-evoked contraction in the first concentration-response curve in each tissue.
Electrically evoked contractions of the guinea pig ileum.
Guinea pigs were killed by a blow on the head. The distal ileum was
removed at least 10 cm proximal to the cecum, and the longitudinal
muscles with the myenteric plexus (2–3 cm in length) were prepared.
The preparations were set up in a 10-ml organ bath containing
Krebs-Henseleit solution. The solution was maintained at 37°C and
saturated with 95% O2 and 5% CO2. A tension of 1 g was applied, and
the response was recorded isometrically through a force displacement transducer. The preparations were suspended between two
parallel platinum wire electrodes and stimulated at supramaximal
voltage with 0.2-Hz square-wave pulses (1 msec in duration) from an
electrical stimulator (SEN-1101, Nihon Kohden Kohgyo). According
to the method of Craig and Clarke (1990), the preparation was
incubated with phenoxybenzamine (3 3 1027 M) for 30 min and
washed out several times. Thereafter, the stimulus voltage was
adjusted to the submaximally effective voltage that induced approximately 50% of the maximal twitch response (approximately 20–35
V). After obtaining stable responses, we applied 5-HT or agonists
cumulatively by increasing the concentration of the drugs, without
washing between concentrations. For cumulative dosing, a new concentration was applied at the moment of maximal effect of the
previous concentration. Only one concentration-response curve was
constructed for each preparation. The effect of the test drug was
expressed as percentage recovery of the twitch response.
[3H]-GR113808 binding in guinea pig brain. Guinea pigs were
decapitated and the brain removed and dissected. Tissue was used
immediately.
For the binding assay, as reported by Grossman et al. (1993),
striatal brain tissue was placed in 15 volumes of HEPES buffer (50
mM, pH 7.4, 4°C) and was homogenized in a Teflon homogenizer and
subsequently centrifuged at 48,000 g and 4°C for 10 min. The resulting pellet was resuspended in HEPES buffer to make a homogenate
at 30 mg/ml. All determinations were performed in duplicate. Assay
tubes contained 400 ml HEPES buffer (65 mM, pH 7.4, 4°C); 100 ml
of a solution of either a competing agent (for drug competition studies), 5-HT to give a final concentration of 30 mM (to determine
nonspecific binding) or deionized water (for determination of total
binding); 400 ml of [3H]-GR113808 in HEPES buffer (50 mM pH 7.4,
4°C) and 100 ml of tissue preparation. [3H]-GR113808 in HEPES
buffer was used to give final concentrations of 0.004 to 1.5 nM for
saturation analysis and 0.1 nM for drug competition studies.
For both saturation analysis and drug competition studies, assay
tubes were incubated at 37°C for 30 min, and the reaction was
terminated by rapid vacuum filtration and washout (3 3 4 ml) with
ice-cold Tris-HCl buffer (pH 7.7) through Whatman GF/B filter paper
using a Brandel Cell Harvester (Brandel Inc. MB-48, Gaithersburg,
MD). Filters were presoaked in a solution of polyethylenimine (0.1%)
to reduce filter binding. Filters were shaken vigorously with 10 ml of
ACS-II scintillation cocktail, and the radioactivity in the filters were
counted using a scintillation counter (ACS-II, Amersham-Japan Int’l
PLC, Tokyo, Japan).
Statistics. Differences from the control group that were statistically significant were identified by means of the YUKMS statistical
analysis program (Yukms Co., Tokyo, Japan) using Student’s t test
for paired data (motor index). The EC50 (relaxation in esophagus,
contraction in distal colon and electrically evoked contraction in
ileum) and IC50 (binding in brain) values, the concentrations causing
50% of the maximum observed for each response, were determined
by linear regression analysis. The pA2 value and slope were calculated by the method of Arunlakshana and Schild (1959).
Vol. 283
1997
In Vitro Studies
Effects of 5-HT4 receptor agonists on carbachol-induced contractions of rat thoracic esophageal muscularis mucosae. A submaximal concentration of 3 mM carba-
Fig. 2. Effect of i.v. administered mosapride, cisapride, zacopride and
BIMU 8 on colonic motor activity in the postprandial state in conscious
dogs. The motor index for the 30-min period after i.v. administration of
test drugs is expressed as a percentage of that for the 30-min period
before administration. Each point represents the mean with the S.E.M.
in 4 to 5 dogs. *P , .05; **P , .01, significant differences from the
solvent treatment. (Student’s t test).
1003
chol produced a well-maintained contraction for at least 60
min. The cumulative addition of 5-HT to the carbachol-contracted rat esophagus caused a concentration-dependent relaxation with an EC50 value of 5.21 6 0.52 nM (n 5 24). The
maximal relaxation with 5-HT occurred at 1 mM. Mosapride,
cisapride, zacopride and BIMU 8 also caused a concentrationdependent relaxation with EC50 values of 208.4 6 33.8 (n 5
6), 39.1 6 3.4 (n 5 6), 317.2 6 106.9 (n 5 8) and 31.5 6 3.8
(n 5 4) nM, respectively, although they were less potent
agonists than 5-HT and were markedly slower to obtain
steady state than 5-HT (fig. 7; table 1). Compared with 5-HT,
these agents behaved as full agonists. Addition of GR113808
(1029 to 1027 M) to the bathing medium did not cause relaxation of the esophagus preparation. GR113808 (1029 to 1027
M) caused a parallel rightward displacement of the concentration-response curve to 5-HT and mosapride (data not
shown). Estimates of the pA2 values for GR113808 against
5-HT and mosapride are 8.85 6 0.06 (Schild plots slope,
1.28 6 0.05) and 8.72 6 0.28 (Schild plots slope, 0.83 6 0.09),
respectively.
Effects of 5-HT4 receptor agonists on the contractions of isolated guinea pig distal colon. In the guinea
pig distal colon, 5-HT evoked monophasic concentration-dependent contractions with an EC50 value of 13.8 6 2.1 nM
(n 5 10). Cisapride and zacopride were full agonists with
EC50 values of 31.5 6 2.9 (n 5 5) and 265 6 39.0 (n 5 6) nM,
respectively. Mosapride and BIMU 8 were partial agonists
relative to 5-HT with intrinsic activities of 0.82 and 0.69,
respectively. The EC50 values of mosapride and BIMU 8 were
estimated to be 3029 6 591 (n 5 13) and 175 6 37.9 (n 5 11)
nM, respectively (fig. 8; table 1).
In the presence of increasing concentrations of GR113808,
the concentration-response curves to 5-HT and mosapride
were displaced to the right in a concentration-dependent
manner (data not shown). The estimate of the pA2 value for
GR113808 against 5-HT was 10.17 6 0.17 (Schild plots slope,
1.15 6 0.16). However, rightward displacements of the concentration-response curve to mosapride were associated with
a reduction in the maximal response with a concentration
ratio of 3.16 yielding estimated pKB values of 9.20 6 0.19.
Effects of 5-HT4 receptor agonists on electrically
evoked contractions of isolated guinea pig ileum.
When cumulatively applied, 5-HT concentration-relatedly
enhanced the twitch response to electrical field stimulation
in guinea pig ileum with an EC50 value of 8.6 6 3.7 (n 5 5)
nM. Other 5-HT4 receptor agonists also enhanced the twitch
response. The EC50 values of mosapride, cisapride, zacopride
and BIMU 8 were 73.2 6 28.3 (n 5 5), 47.6 6 15.6 (n 5 6),
69.0 6 21.4 (n 5 8) and 66.3 6 10.2 (n 5 4) nM, respectively
(table 1). Zacopride was a full agonist, whereas mosapride,
cisapride and BIMU 8 were partial agonists related to 5-HT
with intrinsic activities of 0.58, 0.78 and 0.49, respectively.
Effects of 5-HT4 receptor agonists on [3H]-GR113808
binding in guinea pig brain homogenate. The equilibration saturation studies on the specific binding of [3H]GR113808 revealed a single saturable site of high affinity in
homogenates of guinea pig striatum (Kd 5 0.162 6 0.005 nM,
Bmax 5 62.5 6 1.9 fmol/mg protein). Nonspecific binding
increased linearly with increasing ligand concentration.
5-HT displaced [3H]-GR113808 binding to the 5-HT4 receptors in a concentration-dependent manner with an IC50 value
of 35.8 6 3.3 nM. Mosapride, cisapride, zacopride and BIMU
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mg/kg) lasted longer than that of mosapride (3 mg/kg). Even
at high dose (3 mg/kg), mosapride did not affect the colonic
motor activity during the measurement of GI motor activity.
However, cisapride (0.1–1 mg/kg), zacopride (0.1–1 mg/kg)
and BIMU 8 (0.1–1 mg/kg) stimulated both antral and colonic
motor activity (figs. 1 and 2). Figures 3 and 4 show the typical
tracings of the effects of mosapride, cisapride, zacopride and
BIMU 8. The stimulating effects of these drugs on colonic
motor activity were characterized by GMC-like patterns that
have large-amplitude contractions with rapid propagating
velocity. The effects of cisapride (1 mg/kg) and zacopride (1
mg/kg) were maintained for 60 min, whereas the effect of
BIMU 8 (1 mg/kg) disappeared immediately (figs. 3 and 4). In
addition, some dogs examined defecated after the i.v. administration of cisapride (1 mg/kg) and zacopride (1 mg/kg).
Effects of mosapride and cisapride on antral and
colonic motor activity under treatment with GR113808
in conscious dogs. As shown in figures 5 and 6, GR113808
alone (1 mg/kg i.v., thereafter 1 mg/kg/hr i.v. infusion for 10
min), a selective 5-HT4 receptor antagonist, did not affect
either basal antral or colonic motor activity. Mosapride (3
mg/kg i.v.) and cisapride (1 mg/kg i.v.), when administered 10
min after the start of GR113808 infusion, did not stimulate
either antral or colonic motor activity under treatment with
GR113808 (figs. 5 and 6). The enhanced antral or colonic
motor activity induced by these drugs was antagonized by
treatment with GR113808 in dogs.
Mosapride Stimulates Upper GI Motility
1004
Mine et al.
Vol. 283
Fig. 3. Typical tracings of the effect of
mosapride (panel A) and cisapride (panel
B) on antral and colonic motor activity in
the postprandial state in conscious
dogs. Mosapride (3 mg/kg i.v.) or cisapride (1 mg/kg i.v.) was administered at
the point indicated by the arrow.
8 also inhibited the specific binding of [3H]-GR113808 with
IC50 values of 113 6 14 (n 5 3), 23.0 6 3.1 (n 5 3), 197 6 12
(n 5 3) and 12.6 6 0.9 (n 5 3), respectively (table 1).
Discussion
The present study demonstrates that mosapride selectively
enhanced the upper GI motility in conscious dogs through the
stimulation of 5-HT4 receptors. To clarify this selectivity
further, we compared the effects of mosapride with those of
other existing 5-HT4 receptor agonists— cisapride, zacopride
and BIMU 8 — on antral and colonic motor activity in conscious dogs.
Colonic motor activity in dogs consists of rhythmically
occurring contractile states that have been called migrating
and nonmigrating motor complexes. The main propulsive
force in the canine colon appears to be a function of largeamplitude GMCs. It is well known that GMCs occur during
defecation and are associated with mass movement (Karaus
and Sarna, 1987; Shibata et al., 1993). In the present study,
cisapride, zacopride and BIMU 8 administered by the i.v.
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Fig. 4. Typical tracings of the effect of
zacopride (panel A) and BIMU 8 (panel B) on
antral and colonic motor activity in the postprandial state in conscious dogs. Zacopride
(1 mg/kg i.v.) or BIMU 8 (1 mg/kg i.v.) was
administered at the point indicated by the
arrow.
1997
Mosapride Stimulates Upper GI Motility
1005
Fig. 5. Typical tracings of the effect of
cisapride on antral and colonic motor
activity under treatment with GR113808
in the postprandial state in conscious
dogs. Cisapride (1 mg/kg i.v.) was given
10 min after GR113808 administration (1
mg/kg i.v., thereafter 1 mg/kg/hr infusion
for 20 min).
Fig. 6. Effect of mosapride and cisapride on antral and colonic motor
activity under treatment with GR113808 in the postprandial state in
conscious dogs. A) Effect of mosapride on antral motor activity. B)
Effect of cisapride on antral and colonic motor activity. Mosapride (3
mg/kg i.v.) or cisapride (1 mg/kg i.v.) was given 10 min after GR113808
administration (1 mg/kg i.v., thereafter 1 mg/kg/hr infusion for 20 min).
The control motor index during a 10-min period was expressed as
100%. The second (GR113808 alone) and third (mosapride alone and
its combination with GR113808) indices were expressed as a percentage of the control motor index. Each point represents the mean with the
S.E.M. in 3 to 5 dogs. *P , .05; **P , .01, significant differences from
the solvent treatment. (Student’s t test).
route caused the GMC-like patterns, which have large-amplitude contractions with rapid propagating velocity. In addition, these drugs caused the defecation associated with the
occurrence of GMC-like patterns in some dogs. These observations with cisapride were consistent with those obtained by
Lee et al. (1984) and by Sarna and Otterson (1992). However,
the colonic motility-stimulating effects of cisapride, zacopride
and BIMU 8 had a short duration of action. In contrast, these
drugs at doses 5 to 10 times lower than those that stimulated
colonic motility produced strong and long-lasting contractions in gastric antrum, which indicates a greater sensitivity
in antrum than in colon. On the other hand, mosapride even
at doses 10 times higher than those that enhanced antral
motility failed to produce colonic contractions in any animals.
Accordingly, these comparative data in dogs with four 5-HT4
receptor agonists indicate that mosapride selectively enhances the antral motility and that the other 5-HT4 receptor
agonists enhance both antral and colonic motility. Our findings of different effects on colonic motility among 5-HT4
receptor agonists suggest that the 5-HT4 receptors involved
in colonic motility may be different from those involved in
antral motility.
We have previously reported that the antral motor activity-stimulating effect of mosapride in dogs is decreased after
desensitization of 5-HT receptors, which suggests that 5-HT
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Fig. 7. Cumulative concentration-effect curves to 5-HT, mosapride,
cisapride, zacopride and BIMU 8 in rat esophageal tunica muscularis
mucosae. Each point is the mean of 4 to 24 observations calculated as
a percentage of the control 5-HT maximum.
1006
Mine et al.
Vol. 283
TABLE 1
Summary of comparative agonist potency in the rat esophagus, guinea pig ileum, guinea pig distal colon and guinea pig striatum
[3H]GR113808 Binding of
Guinea Pig Brain
Mosapride
Cisapride
Zacopride
BIMU 8
5-HT
Relaxation of Rat Esophageal
Muscularis Mucosea
Electrically Evoked
Contraction of Guinea Pig
Ileum
Contraction of Guinea Pig Colon
IC50 (nM)
n
EC50 (nM)
n
EC50 (nM)
n
EC50 (nM)
n
112.9 6 13.8
23.0 6 3.1
196.7 6 11.9
12.6 6 0.9
35.8 6 3.3
3
3
3
3
3
208.4 6 33.8
39.1 6 3.4
317.2 6 106.9
31.5 6 3.8
5.2 6 0.5
6
6
8
4
24
73.2 6 28.3
47.6 6 15.6
69.0 6 21.4
66.3 6 12.0
8.6 6 3.7
5
6
8
4
5
3028.7 6 591.0
31.5 6 2.9
265.0 6 39.0
174.7 6 37.9
13.8 6 2.1
13
5
6
11
10
Values are expressed as mean 6 S.E.M.
receptors are involved in the stimulating effect of mosapride
(Yoshida et al., 1991). In addition, the high-dose infusion of
tropisetron, a nonselective 5-HT4 receptor antagonist, was
found to antagonize the stimulating effect of mosapride on
antral motor activity (Yoshida and Itoh, 1994). However, in
in vitro studies, tropisetron has been reported to possess high
5-HT3 receptor antagonistic activity and direct ion channel
and muscarinic cholinergic blocking activity in addition to
the 5-HT4 receptor antagonistic activity (Scholtysik et al.,
1988; Bockaert et al., 1992). We therefore felt it necessary to
examine the interaction between mosapride and more selective 5-HT4 receptor antagonists.
Recently, potent and selective 5-HT4 receptor antagonists
have been introduced, such as SDZ 205-557 (Buchheit et al.,
1991, 1992), DAU 6285 (Turconi et al., 1991; Dumuis et al.,
1992; Schiavone et al., 1992), GR113808 (Grossman et al.,
1993; Gale et al., 1994) and GR12547, SB204070 and
RS39604 (Eglen et al., 1995). Therefore, we decided to investigate whether a highly selective and potent 5-HT4 receptor
antagonist, GR113808, can antagonize the gastroprokinetic
action of mosapride. In the present experiments, GR113808
alone, when infused, did not affect the antral motor activity
in dogs. Under these conditions, the infusion of GR113808
was found to antagonize the stimulating effects of mosapride
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2016
Fig. 8. Concentration-effect curves to 5-HT, mosapride, cisapride,
zacopride and BIMU 8 in guinea pig distal colon LMMP. Experiments
were carried out in the presence of methiothepin (100 nM) and granisetron (1 mM). Each point is the mean of 5 to 13 observations calculated
as a percentage of the control 5-HT maximum.
on antral motor activity in dogs. Similarly, Rizzi et al. (1994)
reported that cisapride and BIMU 1, but not ondansetron,
accelerated gastric emptying of a liquid meal in conscious
dogs and that these effects could be blocked by DAU 6285, another 5-HT4 receptor antagonist. Furthermore, Fankhauser
et al. (1994) demonstrated that SDZ 205-557 inhibited the
stimulatory effect of zacopride on antral, duodenal and jejunal myoelectrical activity in anesthetized dogs. These reports
and our data indicate that the mechanism underlying the
stimulation of gastric and small intestinal motility in dogs by
prokinetic agents is 5-HT4 receptor activation that involves
cholinergic nerves. Previously, there was little evidence that
the colonic motility-stimulating effects of prokinetic agents
could be mediated by activation of 5-HT4 receptors. In the
present study, GR113808 antagonized the stimulating effect
of cisapride on colonic as well as antral motility in dogs.
These findings strongly suggested that the stimulation of GI
motility by prokinetic 5-HT4 receptor agonist is derived from
the 5-HT4 receptor activation, confirming the existence of
5-HT4 receptors in the canine GI tract. However, the mechanism underlying the different profile of the effect of mosapride and other 5-HT4 receptor agonists on colonic motility
remains unclear at present.
To confirm the lack of effect of mosapride on canine colonic
motility further, we compared the in vitro effects of mosapride using guinea pig colon and ileum and rat esophagus
with those of cisapride, zacopride and BIMU 8. In addition,
we investigated the affinity of these drugs for 5-HT4 receptors using [3H]GR113808 ligand. In the present experiments,
5-HT and 5-HT4 receptor agonists caused relaxation of the
carbachol-precontracted rat esophagus, stimulated electrically evoked contractions of guinea pig isolated ileum, induced contractions of guinea pig isolated distal colon and
displaced [3H]GR113808 at the binding sites in guinea pig
striatum homogenates. The responses of four different preparations to these drugs are well documented, and it is widely
accepted that these responses are mediated largely via 5-HT4
receptors (Craig and Clarke, 1990; Baxter et al., 1991; Grossman et al., 1993; Wardle and Sanger, 1993).
As summarized in table 1, the estimated EC50 values of
5-HT, cisapride, zacopride and BIMU 8 correlated well with
functional data on three GI preparations and binding affinities. The rank order of these reference drugs in each model
was also consistent with previous reports (Craig and Clarke,
1990; Baxter et al., 1991; Turconi et al., 1991; Wardle and
Sanger, 1993). Interestingly, this study revealed unexpected
GI tissue selectivity to mosapride. Specifically, mosapride
had a low potency in guinea pig colon compared with the
other GI tissue and binding affinities. The potency of mosa-
1997
1007
In conclusion, the results of this study show that mosapride selectively stimulated upper GI motility in vivo and in
vitro compared with other 5-HT4 receptor agonists. They also
indicate that these different effects of 5-HT4 receptor agonists on GI motility may be based on the existence of heterogeneity of 5-HT4 receptors in the GI tract.
Acknowledgments
The authors thank Dr. T. Karasawa for helpful discussions and
Dr. S. Kato, T. Morie and Dr. H. Harada for synthesizing mosapride,
BIMU 8, zacopride and GR113808.
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