European Journal of Pharmacology, 55 (1979) 181--187
181
© Elsevier]North-Holland Biomedical Press
EFFECTS OF LOPERAMIDE ON THE GUINEA PIG TAENIA COLI IKUNOBU MURAMATSU, MOTOHATSU FUJIWARA, TADAHIRO SHIKIMI * and MIEKO NAKASHIMA *
Department o f Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, and • Department of Pharmacology, Shimane Medical University, Izumo, Shimane 693, Japan Received 24 July 1978, revised MS received 6 December 1978, accepted 11 January 1979
I. MURAMATSU, M. FUJIWARA, T. SHIKIMI and M. NAKASHIMA, Effects of loperamide on the guinea pig taenia coli, European J. Pharmacol. 55 (1979) 181--187. The effects of an antidiarrhoeal agent, loperamide, were studied using isolated guinea pig taenia coli. Loperamide in concentrations ranging from 10 -7 M to 10 -6 M inhibited the cholinergic, contractile responses induced by electrical transmural stimulation, nicotine and serotonin, and the nonadrenergic, relaxing responses induced by electrical transmural stimulation and nicotine. However, the adrenergic response to perivascular nerve stimulation was not affected by these concentrations. The inhibitory effects of loperamide were not reversed by washing with a drug-free solution. Morphine (10 -6 M to 10 -5 M) also inhibited both cholinergic and nonadrenergic responses, but the effect was reversible. Naloxone (10-6 M) attenuated the inhibitory effects of both drugs. Unlike morphine, loperamide in concentrations higher than 5 × 10 -4 M relaxed the strips and reduced the contractile response to acetylcholine noncompetitively, and these effects were not blocked by naloxone. These results suggest that loperamide at low concentrations acts selectively on the opiate receptors located in both cholinergic and nonadrenergic nerves and at higher concentrations also acts directly on smooth muscle thus producing relaxation of the intestinal tone. Nonadrenergic nerve Cholinergic nerve
Opiate receptor
Taenia coli
1. Introduction Loperamide is a new antidiarrhoeal compound, which effectively inhibits both experimental and clinical diarrhoea (Karium and Adaikan, 1977; Heel et al., 1978). At low concentrations the drug inhibits peristaltic activity and the responses of the guinea pig ileum to transmural stimulation and to nicotine (Van Nueten et al., 1974}. At much higher concentrations, it also inhibits the contractions due to histamine, prostaglandins and acetylcholine (Karium and Adaikan, 1977; Van Nueten et al., 1974). Recently, Mackerer et al. (1976) and Wiister and Herz (1978) have shown that loperamide inhibited 3Hnaloxone binding to homogenates of guinea pig and rat brain and of guinea pig myenteric plexus in the same manner as did morphine,
Loperamide
Naloxone
and suggested that the antidiarrhoeal activity of loperamide may be associated with its binding to opiate receptor sites of the intestine. Opiate receptors are widely distributed in the peripheral autonomic nervous system and through such receptors morphine inhibits cholinergic responses in the guinea pig ileum (Gaddum et al., 1957; Paton, 1957; Mackerer, 1976), adrenergic responses in the cat nictitating membrane (Trendelenburg, 1957; Cairnie et al., 1961; Henderson et al., 1975) and mouse vas deferens (Henderson et al., 1972). In the present work, we examined the effects of loperamide on the responses induced by stimulation of cholinergic, adrenergic and nonadrenergic nerves in the guinea pig taenia coli with special reference to opiate receptors.
182 2. Materials and methods Guinea pigs of either sex, weighing about 400 g, were exsanguinated from the c o m m o n carotid arteries and the taenia coli was removed. Strips 1.5 mm in width and 3 mm in length were m o u n t e d vertically in a muscle bath of 20 ml capacity containing the nutrient solution. The initial tension of 1.0 g was loaded and a 90-min equilibration period was allowed before the start of experiment. Changes in isometric tension were recorded through a force-displacement transducer on an ink-writing oscillograph. The bathing media were maintained at 37-+ 0 . 5 ° C and aerated with 95% 02--5% CO2. The composition of the nutrient solution was as follows (mM): NaC1, 154; KCI, 5.4; CaC12 • 2H20, 2.2; NaHCO3, 5.9 and glucose 11.1. The pH of the solution was 7.2. Electrical transmural and perivascular nerve stimulation was performed according to the m e t h o d described by Burnstock et al. (1966). To stimulate the cholinergic and nonadrenergic nerves, square wave pulses of 0.3 msec duration were applied transmurally at frequencies of 10 Hz or 0.5--2.0 Hz for 10 sec. Adrenergic nerves were stimulated perivascularly by using square wave pulses of 1 msec duration at a frequency of 10 Hz for 10 sec. The stimulus voltage was supramaximal. Drugs used were: acetylcholine hydrochloride (ACh), atropine sulfate, morphine hydrochloride, naloxone hydrochloride, nicotine, phentolamine mesylate, propranolol hydrochloride, serotonin creatinine sulfate (5-HT) and t e t r o d o t o x i n (TTX).
3. Results
3.1. Effects o f loperamide on resting tension and A Ch-induced contraction When loperamide (10-~--10 -s M) was added cumulatively to the bath, a relaxing response was elicited with concentrations
I. MURAMATSU ET AL. higher than 10 -6 M. This relaxation was not reversed by washing with a drug-free solution. Propranolol (10 -6 M), phentolamine (10 -6 M), TTX (10 -7 M) and naloxone (10 -6 M) did not affect the relaxing response. Fig. 1A and B show the dose--response curve to ACh and the effects of pretreatment with loperamide and morphine on the ACh response. Loperamide (10 -6 M) did not significantly affect the contractile response to low concentrations of ACh and slightly depressed the response to higher concentrations of ACh. When a high concentration (10 -s M) of loperamide was given, the dose-response curve of ACh shifted to the right and downwards. Morphine (10 -6M, 10 -s M) affected neither the resting tension nor the ACh-induced contraction.
3.2. Effects o f loperamide on the responses to transmural and perivascular nerve stimulation Electrical transmural stimulation produced three types of response; contraction, contraction followed by relaxation and relaxation. The contractile response was abolished by t r e a t m e n t with atropine (10 -6 M) and reversed to one of relaxation. In contrast, the relaxation was enhanced by atropine (10 -6 M), but was not affected by propranolol (10 -6 M) or phentolamine (10 -6 M). TTX (10 -7 M) completely blocked both contractile and relaxing responses, suggesting that the contraction was mediated through cholinergic nerves and the relaxation through nonadrenergic nerves, as reported by Burnstock et al. (1966) and Burnstock (1972). Fig. 2 shows representative results of the effects of loperamide on such responses. Preparation B was pretreated with 10-6M atropine in order to inhibit the cholinergic contractile response. Loperamide (10 -6 M) rapidly reduced both the contractile (A) and the relaxing (B) responses induced by transmural stimulation. Further addition of 10 -6 M naloxone completely restored the inhibitory effects of loperamide. Fig. 3. summarizes the effects of various
LOPERAMIDE ONTAENIACOLI
183
B
A
looi
O
A , lope
"
100
• * control o,morphine lO-eM ~ : morphine lO-SM
-
// /// ]-///
I: O ,i.-. b •*-, co
50
7
8
cone.
5
of
ACh
4
{--looM
3
I
I
I
i
I
I
8
7
0
S
4
3
)
cone. of ACh ( --log M )
Fig. 1. Effects o f l o p e r a m i d e (A) and m o r p h i n e (B) on the contractile responses to acetylcholine (ACh) in guinea pig taenia coll. The c o n t r a c t i o n induced by 2 × 10 -3 M ACh was taken as 100%: m e a n value of the c o n t r a c t i o n to ACh was 2.07 +- 0.47 g. Each value in the figure is the m e a n -+ S.E. of 5 experiments. 2min
A
T
naloxone
IO-SM
--
m,n
L naloxone IO"°M _lg
Iopezamide 10-6M Fig. 2. Effects o f l o p e r a m i d e o n the responses to transmural stimulation (A, B) and perivascular nerve stimulation (C) in guinea pig taenia coli. A: Transmural stimulation (0.3 msec, 10 Hz, 10 sec). B: Transmurai stimulation (0.3 msec, 1 Hz, 10 sec). C: Perivascular nerve stimulation (1 msec, 10 Hz, 10 sec). In B, the responses were recorded under t r e a t m e n t with a t r o p i n e 10-6 M.
184
I. M U R A M A T S U ET AL.
100-
5o-
o; 10-7
5x10-'
t0 "$
conc. of I o p e r a m i d e
(M)
Fig. 3. I n h i b i t i o n b y l o p e r a m i d e o f t h e c o n t r a c t i l e a n d relaxing r e s p o n s e s t o t r a n s m u r a l s t i m u l a t i o n in guined pig t a e n i a coli. The ordinate r e p r e s e n t s t h e p e r c e n t r e s p o n s e relative t o t h e r e s p o n s e in c o n t r o l media. Mean values o f c o n t r o l , c o n t r a c t i l e a n d relaxing r e s p o n s e s w e r e 0.43 ± 0.05 g a n d 0.49 _+ 0.12 g, r e s p e c t i v e l y . E a c h value in t h e figure is t h e m e a n -+ S.E. o f 4 - - 6 e x p e r i m e n t s . Significantly d i f f e r e n t f r o m control, * P < 0.05, * * * P < 0.001. ~, C o n t r a c t i o n ; ~, relaxation.
concentrations of loperamide on such responses. The inhibitory action was roughly dose-dependent; at a concentration of 5 × 10 -6 M of loperamide, the contraction was strongly inhibited and relaxation was inhibited by about 40%. The inhibitory effects of low concentrations o f loperamide (10 -7 and 10 -6 M) were antagonized by the addition of
naloxone (10 -6 M), but the effects of concentrations higher than 5 × 10 -6 M were not antagonized by naloxone (10 -6 and 10 -s M). Table 1 shows the inhibitory effects of 10 -6 M loperamide and the antagonism by naloxone (10 -6 M). Morphine (10 - 6 - 1 0 -s M) also attenuated such contractile and relaxing responses and the inhibition was blocked by 10 -6 M naloxone. Perivascular nerve stimulation produced transient relaxation. Loperamide (fig. 2C) and morphine did n o t significantly affect the relaxing response, but combined treatment with propranolol (10 -6 M) and phentolamine ( 1 0 -6 M), or TTX (10 -7 M) abolished the response. 3.3. Effects o f loperamide on nicotine- or 5-HT-induced responses
Nicotine (10 -4 M) produced either contraction or relaxation, according to the preparations used. The contraction was abolished by treatment with atropine (10 -6 M). However, the relaxation was enhanced by atropine and was not affected by the combined treatment with phentolamine adn propranolol. Fig. 4 summarizes the effects of loperamide on the nicotine-induced responses. The relaxing response was examined after application of
TABLE 1 E f f e c t s o f l o p e r a m i d e and n a l o x o n e o n t h e r e s p o n s e s t o t r a n s m u r a l s t i m u l a t i o n (TS), n i c o t i n e and 5-HT in guinea pig taenia coll. C o n c e n t r a t i o n o f b o t h l o p e r a m i d e and n a l o x o n e was 10 -6 M. Stimulus
Response
n
% Response 1
p :
Loperamide
Loperamide + naloxone
TS
Contraction Relaxation
5 4
57 + 38 +
8 4
95 + 8 81 -+ 7
`(0.01 `(0.01
N i c o t i n e 10 -4 M
Contraction Relaxation
4 8
43 + 15 42 + 5
98 + 13 83 + 3
"(0.05 `(0.001
5-HT 10 -s M
Contraction
4
73 +
97 +- 7
~0.05
6
i E x p r e s s e d as % o f t h e r e s p o n s e s in t h e a b s e n c e o f drugs. 2 Significant d i f f e r e n c e b e t w e e n t h e r e s p o n s e s in t h e a b s e n c e and the p r e s e n c e o f n a l o x o n e ( S t u d e n t ' s t-test).
185
L O P E R A M I D E O N T A E N I A COLI 100-
£
SO-
1 0- I
t 0 -~ conc.
of
Ioperamide
SxlO -e
(M)
Fig. 4. Inhibition by loperamide of the contractile and relaxing responses to nicotine (10 -4 M) in guinea pig taenia coli. The ordinate represents the percent response relative to the control: mean values of the control, contraction and relaxation were 0.55 ± 0,05 g and 0.41 -+ 0.05 g, respectively. Mean -+ S.E. of 5--6 experiments, Significantly different from control, * P < 0.05, * * P < 0.01, * * * P < 0.001. [J, Contraction; m, relaxation.
10-6M atropine to the bath. Loperamide inhibited both responses dose
181
© Elsevier]North-Holland Biomedical Press
EFFECTS OF LOPERAMIDE ON THE GUINEA PIG TAENIA COLI IKUNOBU MURAMATSU, MOTOHATSU FUJIWARA, TADAHIRO SHIKIMI * and MIEKO NAKASHIMA *
Department o f Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, and • Department of Pharmacology, Shimane Medical University, Izumo, Shimane 693, Japan Received 24 July 1978, revised MS received 6 December 1978, accepted 11 January 1979
I. MURAMATSU, M. FUJIWARA, T. SHIKIMI and M. NAKASHIMA, Effects of loperamide on the guinea pig taenia coli, European J. Pharmacol. 55 (1979) 181--187. The effects of an antidiarrhoeal agent, loperamide, were studied using isolated guinea pig taenia coli. Loperamide in concentrations ranging from 10 -7 M to 10 -6 M inhibited the cholinergic, contractile responses induced by electrical transmural stimulation, nicotine and serotonin, and the nonadrenergic, relaxing responses induced by electrical transmural stimulation and nicotine. However, the adrenergic response to perivascular nerve stimulation was not affected by these concentrations. The inhibitory effects of loperamide were not reversed by washing with a drug-free solution. Morphine (10 -6 M to 10 -5 M) also inhibited both cholinergic and nonadrenergic responses, but the effect was reversible. Naloxone (10-6 M) attenuated the inhibitory effects of both drugs. Unlike morphine, loperamide in concentrations higher than 5 × 10 -4 M relaxed the strips and reduced the contractile response to acetylcholine noncompetitively, and these effects were not blocked by naloxone. These results suggest that loperamide at low concentrations acts selectively on the opiate receptors located in both cholinergic and nonadrenergic nerves and at higher concentrations also acts directly on smooth muscle thus producing relaxation of the intestinal tone. Nonadrenergic nerve Cholinergic nerve
Opiate receptor
Taenia coli
1. Introduction Loperamide is a new antidiarrhoeal compound, which effectively inhibits both experimental and clinical diarrhoea (Karium and Adaikan, 1977; Heel et al., 1978). At low concentrations the drug inhibits peristaltic activity and the responses of the guinea pig ileum to transmural stimulation and to nicotine (Van Nueten et al., 1974}. At much higher concentrations, it also inhibits the contractions due to histamine, prostaglandins and acetylcholine (Karium and Adaikan, 1977; Van Nueten et al., 1974). Recently, Mackerer et al. (1976) and Wiister and Herz (1978) have shown that loperamide inhibited 3Hnaloxone binding to homogenates of guinea pig and rat brain and of guinea pig myenteric plexus in the same manner as did morphine,
Loperamide
Naloxone
and suggested that the antidiarrhoeal activity of loperamide may be associated with its binding to opiate receptor sites of the intestine. Opiate receptors are widely distributed in the peripheral autonomic nervous system and through such receptors morphine inhibits cholinergic responses in the guinea pig ileum (Gaddum et al., 1957; Paton, 1957; Mackerer, 1976), adrenergic responses in the cat nictitating membrane (Trendelenburg, 1957; Cairnie et al., 1961; Henderson et al., 1975) and mouse vas deferens (Henderson et al., 1972). In the present work, we examined the effects of loperamide on the responses induced by stimulation of cholinergic, adrenergic and nonadrenergic nerves in the guinea pig taenia coli with special reference to opiate receptors.
182 2. Materials and methods Guinea pigs of either sex, weighing about 400 g, were exsanguinated from the c o m m o n carotid arteries and the taenia coli was removed. Strips 1.5 mm in width and 3 mm in length were m o u n t e d vertically in a muscle bath of 20 ml capacity containing the nutrient solution. The initial tension of 1.0 g was loaded and a 90-min equilibration period was allowed before the start of experiment. Changes in isometric tension were recorded through a force-displacement transducer on an ink-writing oscillograph. The bathing media were maintained at 37-+ 0 . 5 ° C and aerated with 95% 02--5% CO2. The composition of the nutrient solution was as follows (mM): NaC1, 154; KCI, 5.4; CaC12 • 2H20, 2.2; NaHCO3, 5.9 and glucose 11.1. The pH of the solution was 7.2. Electrical transmural and perivascular nerve stimulation was performed according to the m e t h o d described by Burnstock et al. (1966). To stimulate the cholinergic and nonadrenergic nerves, square wave pulses of 0.3 msec duration were applied transmurally at frequencies of 10 Hz or 0.5--2.0 Hz for 10 sec. Adrenergic nerves were stimulated perivascularly by using square wave pulses of 1 msec duration at a frequency of 10 Hz for 10 sec. The stimulus voltage was supramaximal. Drugs used were: acetylcholine hydrochloride (ACh), atropine sulfate, morphine hydrochloride, naloxone hydrochloride, nicotine, phentolamine mesylate, propranolol hydrochloride, serotonin creatinine sulfate (5-HT) and t e t r o d o t o x i n (TTX).
3. Results
3.1. Effects o f loperamide on resting tension and A Ch-induced contraction When loperamide (10-~--10 -s M) was added cumulatively to the bath, a relaxing response was elicited with concentrations
I. MURAMATSU ET AL. higher than 10 -6 M. This relaxation was not reversed by washing with a drug-free solution. Propranolol (10 -6 M), phentolamine (10 -6 M), TTX (10 -7 M) and naloxone (10 -6 M) did not affect the relaxing response. Fig. 1A and B show the dose--response curve to ACh and the effects of pretreatment with loperamide and morphine on the ACh response. Loperamide (10 -6 M) did not significantly affect the contractile response to low concentrations of ACh and slightly depressed the response to higher concentrations of ACh. When a high concentration (10 -s M) of loperamide was given, the dose-response curve of ACh shifted to the right and downwards. Morphine (10 -6M, 10 -s M) affected neither the resting tension nor the ACh-induced contraction.
3.2. Effects o f loperamide on the responses to transmural and perivascular nerve stimulation Electrical transmural stimulation produced three types of response; contraction, contraction followed by relaxation and relaxation. The contractile response was abolished by t r e a t m e n t with atropine (10 -6 M) and reversed to one of relaxation. In contrast, the relaxation was enhanced by atropine (10 -6 M), but was not affected by propranolol (10 -6 M) or phentolamine (10 -6 M). TTX (10 -7 M) completely blocked both contractile and relaxing responses, suggesting that the contraction was mediated through cholinergic nerves and the relaxation through nonadrenergic nerves, as reported by Burnstock et al. (1966) and Burnstock (1972). Fig. 2 shows representative results of the effects of loperamide on such responses. Preparation B was pretreated with 10-6M atropine in order to inhibit the cholinergic contractile response. Loperamide (10 -6 M) rapidly reduced both the contractile (A) and the relaxing (B) responses induced by transmural stimulation. Further addition of 10 -6 M naloxone completely restored the inhibitory effects of loperamide. Fig. 3. summarizes the effects of various
LOPERAMIDE ONTAENIACOLI
183
B
A
looi
O
A , lope
"
100
• * control o,morphine lO-eM ~ : morphine lO-SM
-
// /// ]-///
I: O ,i.-. b •*-, co
50
7
8
cone.
5
of
ACh
4
{--looM
3
I
I
I
i
I
I
8
7
0
S
4
3
)
cone. of ACh ( --log M )
Fig. 1. Effects o f l o p e r a m i d e (A) and m o r p h i n e (B) on the contractile responses to acetylcholine (ACh) in guinea pig taenia coll. The c o n t r a c t i o n induced by 2 × 10 -3 M ACh was taken as 100%: m e a n value of the c o n t r a c t i o n to ACh was 2.07 +- 0.47 g. Each value in the figure is the m e a n -+ S.E. of 5 experiments. 2min
A
T
naloxone
IO-SM
--
m,n
L naloxone IO"°M _lg
Iopezamide 10-6M Fig. 2. Effects o f l o p e r a m i d e o n the responses to transmural stimulation (A, B) and perivascular nerve stimulation (C) in guinea pig taenia coli. A: Transmural stimulation (0.3 msec, 10 Hz, 10 sec). B: Transmurai stimulation (0.3 msec, 1 Hz, 10 sec). C: Perivascular nerve stimulation (1 msec, 10 Hz, 10 sec). In B, the responses were recorded under t r e a t m e n t with a t r o p i n e 10-6 M.
184
I. M U R A M A T S U ET AL.
100-
5o-
o; 10-7
5x10-'
t0 "$
conc. of I o p e r a m i d e
(M)
Fig. 3. I n h i b i t i o n b y l o p e r a m i d e o f t h e c o n t r a c t i l e a n d relaxing r e s p o n s e s t o t r a n s m u r a l s t i m u l a t i o n in guined pig t a e n i a coli. The ordinate r e p r e s e n t s t h e p e r c e n t r e s p o n s e relative t o t h e r e s p o n s e in c o n t r o l media. Mean values o f c o n t r o l , c o n t r a c t i l e a n d relaxing r e s p o n s e s w e r e 0.43 ± 0.05 g a n d 0.49 _+ 0.12 g, r e s p e c t i v e l y . E a c h value in t h e figure is t h e m e a n -+ S.E. o f 4 - - 6 e x p e r i m e n t s . Significantly d i f f e r e n t f r o m control, * P < 0.05, * * * P < 0.001. ~, C o n t r a c t i o n ; ~, relaxation.
concentrations of loperamide on such responses. The inhibitory action was roughly dose-dependent; at a concentration of 5 × 10 -6 M of loperamide, the contraction was strongly inhibited and relaxation was inhibited by about 40%. The inhibitory effects of low concentrations o f loperamide (10 -7 and 10 -6 M) were antagonized by the addition of
naloxone (10 -6 M), but the effects of concentrations higher than 5 × 10 -6 M were not antagonized by naloxone (10 -6 and 10 -s M). Table 1 shows the inhibitory effects of 10 -6 M loperamide and the antagonism by naloxone (10 -6 M). Morphine (10 - 6 - 1 0 -s M) also attenuated such contractile and relaxing responses and the inhibition was blocked by 10 -6 M naloxone. Perivascular nerve stimulation produced transient relaxation. Loperamide (fig. 2C) and morphine did n o t significantly affect the relaxing response, but combined treatment with propranolol (10 -6 M) and phentolamine ( 1 0 -6 M), or TTX (10 -7 M) abolished the response. 3.3. Effects o f loperamide on nicotine- or 5-HT-induced responses
Nicotine (10 -4 M) produced either contraction or relaxation, according to the preparations used. The contraction was abolished by treatment with atropine (10 -6 M). However, the relaxation was enhanced by atropine and was not affected by the combined treatment with phentolamine adn propranolol. Fig. 4 summarizes the effects of loperamide on the nicotine-induced responses. The relaxing response was examined after application of
TABLE 1 E f f e c t s o f l o p e r a m i d e and n a l o x o n e o n t h e r e s p o n s e s t o t r a n s m u r a l s t i m u l a t i o n (TS), n i c o t i n e and 5-HT in guinea pig taenia coll. C o n c e n t r a t i o n o f b o t h l o p e r a m i d e and n a l o x o n e was 10 -6 M. Stimulus
Response
n
% Response 1
p :
Loperamide
Loperamide + naloxone
TS
Contraction Relaxation
5 4
57 + 38 +
8 4
95 + 8 81 -+ 7
`(0.01 `(0.01
N i c o t i n e 10 -4 M
Contraction Relaxation
4 8
43 + 15 42 + 5
98 + 13 83 + 3
"(0.05 `(0.001
5-HT 10 -s M
Contraction
4
73 +
97 +- 7
~0.05
6
i E x p r e s s e d as % o f t h e r e s p o n s e s in t h e a b s e n c e o f drugs. 2 Significant d i f f e r e n c e b e t w e e n t h e r e s p o n s e s in t h e a b s e n c e and the p r e s e n c e o f n a l o x o n e ( S t u d e n t ' s t-test).
185
L O P E R A M I D E O N T A E N I A COLI 100-
£
SO-
1 0- I
t 0 -~ conc.
of
Ioperamide
SxlO -e
(M)
Fig. 4. Inhibition by loperamide of the contractile and relaxing responses to nicotine (10 -4 M) in guinea pig taenia coli. The ordinate represents the percent response relative to the control: mean values of the control, contraction and relaxation were 0.55 ± 0,05 g and 0.41 -+ 0.05 g, respectively. Mean -+ S.E. of 5--6 experiments, Significantly different from control, * P < 0.05, * * P < 0.01, * * * P < 0.001. [J, Contraction; m, relaxation.
10-6M atropine to the bath. Loperamide inhibited both responses dose
