- Email: [email protected]
Effects of atropine, pirenzepine, clonidine, and morphine on biphasic response of rat gastric fundus to field stimulation
~
Pergamon
0306-3623(94)E0023-F
Gen. Pharmac. Vol. 25, No. 5, pp. 951-955, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-3623/94 $7.00 + 0.00
Effects of Atropine, Pirenzepine, Clonidine, and Morphine on Biphasic Response of Rat Gastric Fundus to Field Stimulation AHMAD
REZA
DEHPOUR,* AZIZ DELFAN, KAZEM MOUSAVIZADEH and SEID REZA MORTAZAVI Department o f Pharmacology, School o f Medicine, Tehran University o f Medical Sciences, Tehran, Iran (Received 8 December 1993)
Abstract--1.Electrically evoked contractions in isolated strips of rat
gastric fundus were inhibited by atropine (IC50 = 2.5 x 10 -9 M), pirenzepine (1C50= 2.3 x 10-8 M), clonidine (IC50 = 3.9 x 10 s M) and morphine (IC50 = 3.2 x 10-7 M) in a dose-dependent manner. 2. The inhibitory effect of morphine was antagonized by naloxone (I0 6 M). The inhibitory effect of clonidine not only was not reversed by yohimbine but also was enhanced. Yohimbine per se inhibited these contractions (ICs0 = 6.4 x 10-6 M). 3. In presence of atropine (2 x 10-6 M) and guanethidine (5 x 10 6 M), electrical stimulation of isolated strips of rat gastric fundus produced a non-adrenergic, non-cholinergic (NANC) inhibitory response. 4. The NANC inhibitory response was decreased by morphine (10 8 3 x 10 -6 M). In addition, morphine decreased the tone of the muscle. These effects of morphine was antagonized by naloxone (3 x 10-6 M). 5. Clonidine up to 10 -6 M had no influence on the NANC inhibitory response but yohimbine per se (10-7-3 x 10-5 M) blocked it (IC50 = 3 x 10 6 M). 6. These findings indicate that electrically evoked contractions in the rat gastric fundus were mediated by muscarinic receptors. In addition, the NANC inhibitory response in the isolated 5-trips of rat gastric fundus was blocked by morphine and yohimbine.
Key Words:Rat
gastric fundus, atropine, pirenzepine, clonidine, morphine, non-adrenergic, noncholinergic (NANC) system, nitric oxide
INTRODUCTION
O n the other h a n d m o r p h i n e a n d clonidine are k n o w n to modify n e u r o t r a n s m i t t e r release in the cholinergic system t h r o u g h interaction with presynaptic receptors (Szerb, 1982; Y a u et al., 1983; Starke, 1981). M o r p h i n e was reported to depress the N A N C inhibitory response in the guinea-pig taenia coli (Shimo a n d Ishii, 1978) a n d clonidine was found to e n h a n c e the N A N C excitatory response in the guinea-ig urinary bladder ( M u i r a n d Smart, 1983). However, it was reported neither m o r p h i n e n o r clonidine influences the N A N C inhibitory response in the rat gastric fundus (Lefebvre and Bogaert, 1986). The purpose of this study was to evaluate muscarinic receptor function a n d also to study the effect of m o r p h i n e a n d clonidine o n N A N C inhibitory response in the rat gastric fundus.
The gastric fundus has a n excitatory cholinergic a n d inhibitory non-adrenergic, non-cholinergic ( N A N C ) i n n e r v a t i o n (Li a n d R a n d , 1990). There is evidence to suggest that pre-junctional ~2-adrenoceptor mediated the inhibition o f b o t h cholinergic excitatory a n d the N A N C inhibitory nerve-induced responses (Verplanken et al., 1984; D e t t m a r et al., 1984, 1985a, b; M a c D o n a l d et al., 1990). The N A N C neurons have been recognized in m a n y parts of the gastrointestinal tract a n d have recently been linked with release of nitric oxide (NO) on electrical stimulation ( A b r a h a m s s o n , 1986; Li a n d Rand, 1990; Boeckxstaens et al., 1990; Desai et al., 1991).
*To whom all correspondence should be addressed. 951
952
AHMAD REZA
MATERIALS AND M E T H O D S
DEHPOURet al. lo0
In vitro experiments Male Sprague-Dawley rats, 150-300 g, were killed by a blow to the head and cervical dislocation. The gastric fundus was removed and strips prepared by cutting a parallel to the greater curvature. Tissues were suspended between two platinum "ring and hook" electrodes in 20 ml organ baths containing Krebs medium of following composition (mM): NaCI, 118.3; KCI, 4.7; CaC12, 2.5; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; D-glucose, 11.1. The solution was maintained at 36°C and gassed continuously with 5% CO 2 in oxygen. Each muscle was placed under an initial resting tension of 1 g and allowed to equilibrate for 1 hr before any further experimental procedures. Muscle tension was recorded with an isometric transducer and displayed on DMR-4B physiograph (Narco Biosystem Inc.). When the NANC inhibitory responses were investigated, the Krebs solution contained atropine (2 × 10-6 M) and guanethidine (5 × 10 6 M) to block cholinergic and adrenergic in responses to field stimulation of intramural nerves, and then serotonin (5 x 10-7M) was added to raise the tone of the muscle. Electrical stimulation Train electrical stimulation were applied in one of the following manner: 1. When contractions of the muscle were investigated, impulses of 1 ms and supramaximal voltage from a grass $88 stimulator were applied for 8 sec every 120 sec at 10 Hz frequency. 2. When the NANC inhibitory responses were investigated, impulses of 1 msec duration and supramaximal voltage from a grass $88 stimulator were applied for 6 sec every 120 sec at 5 Hz frequency. Statistical analysis The responses of a drug were expressed as percent of response compared with control. Results are given as mean + SEM and the number of experiments as n. IC50 was calculated from dose-response curve.
8O 60
~ 40 21
-10
n=6 I
I
I
-9
-8
-7
I -6
Log [Dose]
Fig. 1. The dose-response curves for the inhibitory effects of pirenzepine (O) and atropine (O) on contractions produced by train field stimulation (supramaximal voltage, I ms duration, 10 Hz, 120 sec) in the rat gastric fundus. Each point is mean of six experiments and the bars represent ___SE.
RESULTS Field stimulation Electrical field stimulation produced rapid and reproducible relaxation and then abruptly contraction in the rat gastric fundus. In presence of atropine (2 × 10 - 6 M) and guanethidine (5 × 10-6 M), electrical stimulation produces only relaxatory response. I. Effects of atropine, pirenzepine, clonidine, and morphine on electrically nerve-induced contractions. Electrically nerve-induced contractions of rat gastric fundus were inhibited by atropine (3 x 10-1°-3 x 10 -8 M) and pirenzepine (3 × 10 - 9 3 × 10 -7 M ) (Fig. 1). IC50 of atropine and pirenzepine was 2.5 × 10-9M, 2.3 x 10-8M, respectively. Atropine not only inhibited the contractions but also enhanced the relaxatory response (Fig. 2). Clonidine (3 x 10-9-3 x 10 -6 M) and morphine (10-8-10 -5 M) inhibited electrically nerve-induced contractions (Fig. 3). IC50 of clonidine and morphine was
2m~Ain
Drugs The following drugs were used: atropine sulphate (Sigma Co.), clonidine hydrochloride (BoehringerIngelheim), guanethidine monosulphate (Sigma Co.), 5-hydroxytryptamine creatinine sulphate (Sigma Co.), morphine hydrochloride (MacFarlan Smith Ltd), naloxone hydrochloride (Sigma Co.), pirenzepine dihydrochloride (Sigma Co.), and yohimbine hydrochloride (Sigma Co.). All drugs were dissolved in deionized water.
Fig. 2. The biphasic effect of atropine (a = 3 × 10 to M, b = 10 -9 M, c = 3 x I0 9M, d = 10-8 M, e = 3 x 10-s M) on contractions produced by train field stimulation in the rat gastric fundus.
Biphasic response of rat gastric fundus 100
953
2rain
80
~o
6O
a
b
c
d
Fig. 5. The inhibitory effect of yohimbine (a = 3 x 10-7 M, b = 10 -6 M, c = 3 × 10 -6 M, d = 10-5 M) on contractions produced by train field stimulation in the rat gastric fundus.
40 6 20
0-10
. 19 .
[8.
.ll7
.
16
15
I4
Log [Dose]
Fig. 3. The dose-response curves for the inhibitoyeffectof morphine (@) and clonidine (O) on contractions produced by train field stimulation in the rat gastric fundus. Each point is mean of six experiments and the bars represent __+SE.
3.9 x 10-SM, 3.2 × 10-TM, respectively. In this experiments, clonidine not only inhibited the contractions but also enhanced the relaxatory response (Fig. 4). The inhibitory effect of morphine was antagonized by naloxone (10 -6 M ) but the inhibitory effect clonidine was not antagonized by yohimbine (not shown in figures). Yohimbine (3 x 10-7-10 -5 M) per se blocked these contractions in a dose dependent manner (Fig. 5). IC50 for this inhibitory effect of yohimbine was 6.4 x 10 -6 M. 2. Effects of morphine and clonidine on N A N C inhibitory response. The N A N C inhibitory response due to electrical stimulation was decreased by morphine (10 -83 x 10 -6 M). Clonidine up to 1 0 - 6 M had no influence on this inhibitory response (not shown in figures), but yohimbine (10-7-3 x 10 -5 M) blocked this response (Fig. 6). IC50 of yohimbine was 3 × 10 -6 M. Both the reduction of N A N C inhibitory response due to electrical stimulation and the
reduction of the tone of the muscle induced by morphine, were reversed by naloxone (3 x 10 -6 M) (Fig. 7). DISCUSSION The present study shows that atropine and pirenzepine inhibited electrically nerve-induced contractions of the rat gastric fundus in a dose-dependent manner. The concentration of pirenzepine required to cause the 50% of inhibition (IC50) was about 10 times greater than those of atropine. This difference corresponds to the difference in antagonistic potencies of pirenzepine and atropine at the different muscarinic receptors of rat gastric fundus. This confirms earlier studies which show that M : r e c e p t o r with a high affinity and M2-receptor with a low affinity to pirenzepine exist on the nerve and smooth muscle of gastric fundus respectively (Hamer, 1980; Hirschowitz et al., 1983). It was reported that pre-junctional ~-adrenoceptor inhibited the cholinergic excitatory nerve-induced response (Verplanken et al., 1984; M a c D o n a l d et al., 1O0 80
o
60
~
4o 20
0 I
I
I
-7
-6
-5
I -4
Log [ D o s e ] a
b
c
d
¢
f
g
Fig. 4. The biphasic effect of clonidine (a = 3 x 10 -9 M, b = I0 -8 M, c = 3 x 10-s M, d = 10 -7 M, e = 3 x 10-7 M, f = 10-6 M, g = 3 x 10 -6 M) on contractions produced by train field stimulation in the rat gastric fundus.
Fig. 6. The dose-response curve for the inhibitory effect of yohimbine on non-adrenergic, non-cholinergic (NANC) response produced by field train stimulation (supramaximal voltage, l ms, 5Hz, 120see) in the rat gastric fundus. Each point is mean of six experiments and the bars represent ___SE.
954
AHMADREZADEI4POURet al.
2min 1 [ "]'~f]f'[/"]/"] ~ ,"n ,"~~
Mor
a
b
c
d
Nal
e
rl/"]/~//]/
f
Fig. 7. The inhibitory effect of morphine (a = 10-8 M, b = 3 x 10-s M, c = 10 -7 M, d = 3 × 10-7 M, e = 10-6 M, f = 3 x 10-6M) and the reversal action of naloxone (3 × 10-6 M) on non-adrenergic, non-cholinergic (NANC) response produced by train field stimulation in the rat gastric fundus.
1990). In this study, we found that, clonidine, ~2adrenoceptor agonist, inhibited the cholinergic nerveinduced contractions in a dose-dependent manner (IC~0=3.9 × 10 8M). Interestingly this effect of clonidine was not reversed by yohimbine. This is in contrast to the finding of Verplanken et al. (1984), who found that the effect of clonidine on electrical stimulation-induced contractions of the muscle was antagonized competitively by yohimbine. The difference may be due to the different stimulation parameters; in the present study the cholinergic nerve-induced response were obtained at 1 0 H z whereas Verplanken et al. (1984) used a lower frequency (1 Hz). The inhibitory response of the muscle in the field stimulation also potentiated by higher concentrations of clonidine (10 -6 M). This is probably due to relaxatory effect of clonidine which mediated by postjunctional ~-adrenoceptors. This results confirms the previous findings of inhibitory effect of clonidine on post-junctional ~-adrenoceptors in the rat gastric fundus (Verplanken et al., 1984; M a c D o n a l d et al., 1990). It can be concluded that in the gastric fundus, clonidine inhibits the cholinergic nerve activity via pre-junctional ~2-adrenoceptors and only at high concentrations (3 x 10 -6 M ) it relaxes the muscle via post-junctional ~-adrenoceptor. The latter result confirmed the previous report by Verplanken et al. (1984). In the present study we were unable to demonstrate any effects of clonidine (up to 10 -6 M ) on the N A N C inhibitory response. This is in contrast to the finding of M a c D o n a l d et al. (1990) and confirmed the previous report of Lefebvre and Bogaert (1986). Yohimbine p e r se abolished both the cholinergic excitatory and the N A N C inhibitory response of the muscle. However these effects were observed in high concentrations (up to 10 -6 M) of the drug and may be to due antagonistic effect of yohimbine on other receptors, including serotonin, dopamine, and ~-receptors (Kramer, 1988). The cholinergic contractions of the rat gastric fundus in response to electrical field stimulation is inhibited by morphine in a dose-dependent manner. The inhibi-
tory effect of morphine was found to be susceptible to blockade by naloxone (10 -6 M), suggesting that opioid receptors are involved in the inhibitory response o f morphine in this preparation. This effect of morphine is probably due to the prevention of acetylcholine release at cholinergic nerve-effector junctions (Szerb, 1982; Yau et al., 1983). Our results show that morphine also decreased the N A N C inhibitory response of the muscle. This is in contrast to the findings of Lefebvre and Bogaert (1986), who found that morphine up to 10-SM did not influence the N A N C oxidation. On the other hand Calignano et al. (1991) have reported that endogenous nitric oxide modulates morphine-induced constipation. It seems that the inhibitory effects of morphine on both cholinergic nerve-induced contractions and the N A N C relaxatory responses of the rat gastric fundus may be due to reduction of acetylcholine release (Kramer, 1988; Gillian and Pollock, 1976). Acknowledgement--The authors would be like to thank Dr
Mahmoud Ghazi Khansari for his advice on the use of the computer in preparing the manuscript.
REFERENCES Abrahamsson H. (1986) Non-adrenergic, non-cholinergic nervous control of gastrointestinal motility patterns. Arch. Int. Pharmacodyn. Ther. 280, 50-60. Boeckxstaens G. E., Pelckmans P. A., Bogers J. J., Bult H., De Man J. G., Ooster bosch L., Herman A. G. and Van Maerk Y. M. (1990) Release of nitric oxide upon stimulation of non-adrenergic, non-cholinergic nerves in the rat gastric fundus. J. Pharmac. Exp. Ther. 256, 441-447. Calignano A., Moncada S. and Di-Rosa M. (1991) Endogenous nitric oxide modulates morphine-induced constipation. Biochem. biophys. Res. Commun. 191, 889-893. Desai K. M., Sessa W. C. and Vane J. R. (1991) Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature 351,477~,79. Dettmar P. W., Kelly J. and MacDonald A. (1984) Characterization of ct-adrenoceptor-mediated response in the rat gastric fundus. Br. J. Pharmacol. 83, p.390. Dettmar P. W., Kelly J. and MacDonald A. (1985a) The effects of UK-14,304 on nerve-induced responses in the rat gastric fundus. Br. J. PharmacoL 80, p.491. Dettmar P. W., Kelly J. and MacDonald A. (198519) Alfa~and alpha.,- adrenoceptor-mediated effects of noradrenaline in the rat gastric fundus. Br. J. Pharmacol. 86, p.613. Gillian M. G. C. and Pollock D. (1976) Investigation of the effects drugs on morphine-induced contraction of the isolated colon of the rat. Br. J. Pharmacol. 57, 444~,45. Hammer R. (1980) Muscarinic receptors in the stomach. Scan. J. Gastroentrol. 66, 5-I1. Hirschowitz B. I., Fong J. and Molina E. (1983) Effect of pirenzepine on vagal and cholinergic gastric secretion and gastrin release and on heart rate in the dog. J. Pharmae, Exp. Ther. 225, 263-268. Kramer W. (1988) Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion. Pharmaeol. Rev. 411, 121-126. Lefebvre R. A. and Bogaert M. G. (1986) Neither morphine nor clonidine influence the non-adrenergic, non-cholinergic inhibitory response in the rat gastric fundus. J. Pharm. Pharmaeol. 38, 621~522.
Biphasic response of rat gastric fundus Lefebvre R. A. (1986) Study on the possible neurotransmitter of the non-adrenergic, non-cholinergic innervation of the rat gastric fundus. Arch. Int. Pharmacodyn. 280, 110. Li. C. G. and Rand M. J. (1990) Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, noncholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur. J. Pharrnac. 4, 191 303-309. MacDonald A., Kelly J. and Dettmar P. W. (1990) Pre- and post-junctional ~t-adrenoceptor mediated response in the rat gastric fundus in vitro. J. Pharrn. Pharmacol. 42, 752-757. Muir T. C. and Smart N. G. (1983) The effects of clonidine on the response to stimulation of nonadrenergic, non-cholinergic nerves in the guinea-pig urinary bladder in vitro. J. Pharm. Pharmacol. 35, 234--237.
955
Shimo Y. and Ishii T. (1978) Effects of morphine on non-adrenergic, inhibitory response of the guinea-pig tania-coli. J. Pharm. Pharmacol. 30, 596-570. Starke K. (1981) ~t-adrenoceptor subclassification. Rev. Physiol. Biochem. Pharmac. 88, 199-236. Szerb J. C. (1982) Correlation between acetylcholine release and neuronal activity in the guinea-pig ileum myenteric plexus; effect of morphine. Neuroscience 7, 327-340. Verplanken P. A., Lefebvre R. A. and Bogaert M. G. (1984) Pharmacological characterization of ct-adrenoceptor in the rat gastric fundus. J. Pharmac. Exp. Ther. 231, 404-410. Yau W. M. and Lingle P. F. (1983) Interaction of enkephaline and caerulein on guinea-pig small intestine. Am. J. Physiol. 244, G65-G70.
Pergamon
0306-3623(94)E0023-F
Gen. Pharmac. Vol. 25, No. 5, pp. 951-955, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-3623/94 $7.00 + 0.00
Effects of Atropine, Pirenzepine, Clonidine, and Morphine on Biphasic Response of Rat Gastric Fundus to Field Stimulation AHMAD
REZA
DEHPOUR,* AZIZ DELFAN, KAZEM MOUSAVIZADEH and SEID REZA MORTAZAVI Department o f Pharmacology, School o f Medicine, Tehran University o f Medical Sciences, Tehran, Iran (Received 8 December 1993)
Abstract--1.Electrically evoked contractions in isolated strips of rat
gastric fundus were inhibited by atropine (IC50 = 2.5 x 10 -9 M), pirenzepine (1C50= 2.3 x 10-8 M), clonidine (IC50 = 3.9 x 10 s M) and morphine (IC50 = 3.2 x 10-7 M) in a dose-dependent manner. 2. The inhibitory effect of morphine was antagonized by naloxone (I0 6 M). The inhibitory effect of clonidine not only was not reversed by yohimbine but also was enhanced. Yohimbine per se inhibited these contractions (ICs0 = 6.4 x 10-6 M). 3. In presence of atropine (2 x 10-6 M) and guanethidine (5 x 10 6 M), electrical stimulation of isolated strips of rat gastric fundus produced a non-adrenergic, non-cholinergic (NANC) inhibitory response. 4. The NANC inhibitory response was decreased by morphine (10 8 3 x 10 -6 M). In addition, morphine decreased the tone of the muscle. These effects of morphine was antagonized by naloxone (3 x 10-6 M). 5. Clonidine up to 10 -6 M had no influence on the NANC inhibitory response but yohimbine per se (10-7-3 x 10-5 M) blocked it (IC50 = 3 x 10 6 M). 6. These findings indicate that electrically evoked contractions in the rat gastric fundus were mediated by muscarinic receptors. In addition, the NANC inhibitory response in the isolated 5-trips of rat gastric fundus was blocked by morphine and yohimbine.
Key Words:Rat
gastric fundus, atropine, pirenzepine, clonidine, morphine, non-adrenergic, noncholinergic (NANC) system, nitric oxide
INTRODUCTION
O n the other h a n d m o r p h i n e a n d clonidine are k n o w n to modify n e u r o t r a n s m i t t e r release in the cholinergic system t h r o u g h interaction with presynaptic receptors (Szerb, 1982; Y a u et al., 1983; Starke, 1981). M o r p h i n e was reported to depress the N A N C inhibitory response in the guinea-pig taenia coli (Shimo a n d Ishii, 1978) a n d clonidine was found to e n h a n c e the N A N C excitatory response in the guinea-ig urinary bladder ( M u i r a n d Smart, 1983). However, it was reported neither m o r p h i n e n o r clonidine influences the N A N C inhibitory response in the rat gastric fundus (Lefebvre and Bogaert, 1986). The purpose of this study was to evaluate muscarinic receptor function a n d also to study the effect of m o r p h i n e a n d clonidine o n N A N C inhibitory response in the rat gastric fundus.
The gastric fundus has a n excitatory cholinergic a n d inhibitory non-adrenergic, non-cholinergic ( N A N C ) i n n e r v a t i o n (Li a n d R a n d , 1990). There is evidence to suggest that pre-junctional ~2-adrenoceptor mediated the inhibition o f b o t h cholinergic excitatory a n d the N A N C inhibitory nerve-induced responses (Verplanken et al., 1984; D e t t m a r et al., 1984, 1985a, b; M a c D o n a l d et al., 1990). The N A N C neurons have been recognized in m a n y parts of the gastrointestinal tract a n d have recently been linked with release of nitric oxide (NO) on electrical stimulation ( A b r a h a m s s o n , 1986; Li a n d Rand, 1990; Boeckxstaens et al., 1990; Desai et al., 1991).
*To whom all correspondence should be addressed. 951
952
AHMAD REZA
MATERIALS AND M E T H O D S
DEHPOURet al. lo0
In vitro experiments Male Sprague-Dawley rats, 150-300 g, were killed by a blow to the head and cervical dislocation. The gastric fundus was removed and strips prepared by cutting a parallel to the greater curvature. Tissues were suspended between two platinum "ring and hook" electrodes in 20 ml organ baths containing Krebs medium of following composition (mM): NaCI, 118.3; KCI, 4.7; CaC12, 2.5; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; D-glucose, 11.1. The solution was maintained at 36°C and gassed continuously with 5% CO 2 in oxygen. Each muscle was placed under an initial resting tension of 1 g and allowed to equilibrate for 1 hr before any further experimental procedures. Muscle tension was recorded with an isometric transducer and displayed on DMR-4B physiograph (Narco Biosystem Inc.). When the NANC inhibitory responses were investigated, the Krebs solution contained atropine (2 × 10-6 M) and guanethidine (5 × 10 6 M) to block cholinergic and adrenergic in responses to field stimulation of intramural nerves, and then serotonin (5 x 10-7M) was added to raise the tone of the muscle. Electrical stimulation Train electrical stimulation were applied in one of the following manner: 1. When contractions of the muscle were investigated, impulses of 1 ms and supramaximal voltage from a grass $88 stimulator were applied for 8 sec every 120 sec at 10 Hz frequency. 2. When the NANC inhibitory responses were investigated, impulses of 1 msec duration and supramaximal voltage from a grass $88 stimulator were applied for 6 sec every 120 sec at 5 Hz frequency. Statistical analysis The responses of a drug were expressed as percent of response compared with control. Results are given as mean + SEM and the number of experiments as n. IC50 was calculated from dose-response curve.
8O 60
~ 40 21
-10
n=6 I
I
I
-9
-8
-7
I -6
Log [Dose]
Fig. 1. The dose-response curves for the inhibitory effects of pirenzepine (O) and atropine (O) on contractions produced by train field stimulation (supramaximal voltage, I ms duration, 10 Hz, 120 sec) in the rat gastric fundus. Each point is mean of six experiments and the bars represent ___SE.
RESULTS Field stimulation Electrical field stimulation produced rapid and reproducible relaxation and then abruptly contraction in the rat gastric fundus. In presence of atropine (2 × 10 - 6 M) and guanethidine (5 × 10-6 M), electrical stimulation produces only relaxatory response. I. Effects of atropine, pirenzepine, clonidine, and morphine on electrically nerve-induced contractions. Electrically nerve-induced contractions of rat gastric fundus were inhibited by atropine (3 x 10-1°-3 x 10 -8 M) and pirenzepine (3 × 10 - 9 3 × 10 -7 M ) (Fig. 1). IC50 of atropine and pirenzepine was 2.5 × 10-9M, 2.3 x 10-8M, respectively. Atropine not only inhibited the contractions but also enhanced the relaxatory response (Fig. 2). Clonidine (3 x 10-9-3 x 10 -6 M) and morphine (10-8-10 -5 M) inhibited electrically nerve-induced contractions (Fig. 3). IC50 of clonidine and morphine was
2m~Ain
Drugs The following drugs were used: atropine sulphate (Sigma Co.), clonidine hydrochloride (BoehringerIngelheim), guanethidine monosulphate (Sigma Co.), 5-hydroxytryptamine creatinine sulphate (Sigma Co.), morphine hydrochloride (MacFarlan Smith Ltd), naloxone hydrochloride (Sigma Co.), pirenzepine dihydrochloride (Sigma Co.), and yohimbine hydrochloride (Sigma Co.). All drugs were dissolved in deionized water.
Fig. 2. The biphasic effect of atropine (a = 3 × 10 to M, b = 10 -9 M, c = 3 x I0 9M, d = 10-8 M, e = 3 x 10-s M) on contractions produced by train field stimulation in the rat gastric fundus.
Biphasic response of rat gastric fundus 100
953
2rain
80
~o
6O
a
b
c
d
Fig. 5. The inhibitory effect of yohimbine (a = 3 x 10-7 M, b = 10 -6 M, c = 3 × 10 -6 M, d = 10-5 M) on contractions produced by train field stimulation in the rat gastric fundus.
40 6 20
0-10
. 19 .
[8.
.ll7
.
16
15
I4
Log [Dose]
Fig. 3. The dose-response curves for the inhibitoyeffectof morphine (@) and clonidine (O) on contractions produced by train field stimulation in the rat gastric fundus. Each point is mean of six experiments and the bars represent __+SE.
3.9 x 10-SM, 3.2 × 10-TM, respectively. In this experiments, clonidine not only inhibited the contractions but also enhanced the relaxatory response (Fig. 4). The inhibitory effect of morphine was antagonized by naloxone (10 -6 M ) but the inhibitory effect clonidine was not antagonized by yohimbine (not shown in figures). Yohimbine (3 x 10-7-10 -5 M) per se blocked these contractions in a dose dependent manner (Fig. 5). IC50 for this inhibitory effect of yohimbine was 6.4 x 10 -6 M. 2. Effects of morphine and clonidine on N A N C inhibitory response. The N A N C inhibitory response due to electrical stimulation was decreased by morphine (10 -83 x 10 -6 M). Clonidine up to 1 0 - 6 M had no influence on this inhibitory response (not shown in figures), but yohimbine (10-7-3 x 10 -5 M) blocked this response (Fig. 6). IC50 of yohimbine was 3 × 10 -6 M. Both the reduction of N A N C inhibitory response due to electrical stimulation and the
reduction of the tone of the muscle induced by morphine, were reversed by naloxone (3 x 10 -6 M) (Fig. 7). DISCUSSION The present study shows that atropine and pirenzepine inhibited electrically nerve-induced contractions of the rat gastric fundus in a dose-dependent manner. The concentration of pirenzepine required to cause the 50% of inhibition (IC50) was about 10 times greater than those of atropine. This difference corresponds to the difference in antagonistic potencies of pirenzepine and atropine at the different muscarinic receptors of rat gastric fundus. This confirms earlier studies which show that M : r e c e p t o r with a high affinity and M2-receptor with a low affinity to pirenzepine exist on the nerve and smooth muscle of gastric fundus respectively (Hamer, 1980; Hirschowitz et al., 1983). It was reported that pre-junctional ~-adrenoceptor inhibited the cholinergic excitatory nerve-induced response (Verplanken et al., 1984; M a c D o n a l d et al., 1O0 80
o
60
~
4o 20
0 I
I
I
-7
-6
-5
I -4
Log [ D o s e ] a
b
c
d
¢
f
g
Fig. 4. The biphasic effect of clonidine (a = 3 x 10 -9 M, b = I0 -8 M, c = 3 x 10-s M, d = 10 -7 M, e = 3 x 10-7 M, f = 10-6 M, g = 3 x 10 -6 M) on contractions produced by train field stimulation in the rat gastric fundus.
Fig. 6. The dose-response curve for the inhibitory effect of yohimbine on non-adrenergic, non-cholinergic (NANC) response produced by field train stimulation (supramaximal voltage, l ms, 5Hz, 120see) in the rat gastric fundus. Each point is mean of six experiments and the bars represent ___SE.
954
AHMADREZADEI4POURet al.
2min 1 [ "]'~f]f'[/"]/"] ~ ,"n ,"~~
Mor
a
b
c
d
Nal
e
rl/"]/~//]/
f
Fig. 7. The inhibitory effect of morphine (a = 10-8 M, b = 3 x 10-s M, c = 10 -7 M, d = 3 × 10-7 M, e = 10-6 M, f = 3 x 10-6M) and the reversal action of naloxone (3 × 10-6 M) on non-adrenergic, non-cholinergic (NANC) response produced by train field stimulation in the rat gastric fundus.
1990). In this study, we found that, clonidine, ~2adrenoceptor agonist, inhibited the cholinergic nerveinduced contractions in a dose-dependent manner (IC~0=3.9 × 10 8M). Interestingly this effect of clonidine was not reversed by yohimbine. This is in contrast to the finding of Verplanken et al. (1984), who found that the effect of clonidine on electrical stimulation-induced contractions of the muscle was antagonized competitively by yohimbine. The difference may be due to the different stimulation parameters; in the present study the cholinergic nerve-induced response were obtained at 1 0 H z whereas Verplanken et al. (1984) used a lower frequency (1 Hz). The inhibitory response of the muscle in the field stimulation also potentiated by higher concentrations of clonidine (10 -6 M). This is probably due to relaxatory effect of clonidine which mediated by postjunctional ~-adrenoceptors. This results confirms the previous findings of inhibitory effect of clonidine on post-junctional ~-adrenoceptors in the rat gastric fundus (Verplanken et al., 1984; M a c D o n a l d et al., 1990). It can be concluded that in the gastric fundus, clonidine inhibits the cholinergic nerve activity via pre-junctional ~2-adrenoceptors and only at high concentrations (3 x 10 -6 M ) it relaxes the muscle via post-junctional ~-adrenoceptor. The latter result confirmed the previous report by Verplanken et al. (1984). In the present study we were unable to demonstrate any effects of clonidine (up to 10 -6 M ) on the N A N C inhibitory response. This is in contrast to the finding of M a c D o n a l d et al. (1990) and confirmed the previous report of Lefebvre and Bogaert (1986). Yohimbine p e r se abolished both the cholinergic excitatory and the N A N C inhibitory response of the muscle. However these effects were observed in high concentrations (up to 10 -6 M) of the drug and may be to due antagonistic effect of yohimbine on other receptors, including serotonin, dopamine, and ~-receptors (Kramer, 1988). The cholinergic contractions of the rat gastric fundus in response to electrical field stimulation is inhibited by morphine in a dose-dependent manner. The inhibi-
tory effect of morphine was found to be susceptible to blockade by naloxone (10 -6 M), suggesting that opioid receptors are involved in the inhibitory response o f morphine in this preparation. This effect of morphine is probably due to the prevention of acetylcholine release at cholinergic nerve-effector junctions (Szerb, 1982; Yau et al., 1983). Our results show that morphine also decreased the N A N C inhibitory response of the muscle. This is in contrast to the findings of Lefebvre and Bogaert (1986), who found that morphine up to 10-SM did not influence the N A N C oxidation. On the other hand Calignano et al. (1991) have reported that endogenous nitric oxide modulates morphine-induced constipation. It seems that the inhibitory effects of morphine on both cholinergic nerve-induced contractions and the N A N C relaxatory responses of the rat gastric fundus may be due to reduction of acetylcholine release (Kramer, 1988; Gillian and Pollock, 1976). Acknowledgement--The authors would be like to thank Dr
Mahmoud Ghazi Khansari for his advice on the use of the computer in preparing the manuscript.
REFERENCES Abrahamsson H. (1986) Non-adrenergic, non-cholinergic nervous control of gastrointestinal motility patterns. Arch. Int. Pharmacodyn. Ther. 280, 50-60. Boeckxstaens G. E., Pelckmans P. A., Bogers J. J., Bult H., De Man J. G., Ooster bosch L., Herman A. G. and Van Maerk Y. M. (1990) Release of nitric oxide upon stimulation of non-adrenergic, non-cholinergic nerves in the rat gastric fundus. J. Pharmac. Exp. Ther. 256, 441-447. Calignano A., Moncada S. and Di-Rosa M. (1991) Endogenous nitric oxide modulates morphine-induced constipation. Biochem. biophys. Res. Commun. 191, 889-893. Desai K. M., Sessa W. C. and Vane J. R. (1991) Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid. Nature 351,477~,79. Dettmar P. W., Kelly J. and MacDonald A. (1984) Characterization of ct-adrenoceptor-mediated response in the rat gastric fundus. Br. J. Pharmacol. 83, p.390. Dettmar P. W., Kelly J. and MacDonald A. (1985a) The effects of UK-14,304 on nerve-induced responses in the rat gastric fundus. Br. J. PharmacoL 80, p.491. Dettmar P. W., Kelly J. and MacDonald A. (198519) Alfa~and alpha.,- adrenoceptor-mediated effects of noradrenaline in the rat gastric fundus. Br. J. Pharmacol. 86, p.613. Gillian M. G. C. and Pollock D. (1976) Investigation of the effects drugs on morphine-induced contraction of the isolated colon of the rat. Br. J. Pharmacol. 57, 444~,45. Hammer R. (1980) Muscarinic receptors in the stomach. Scan. J. Gastroentrol. 66, 5-I1. Hirschowitz B. I., Fong J. and Molina E. (1983) Effect of pirenzepine on vagal and cholinergic gastric secretion and gastrin release and on heart rate in the dog. J. Pharmae, Exp. Ther. 225, 263-268. Kramer W. (1988) Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion. Pharmaeol. Rev. 411, 121-126. Lefebvre R. A. and Bogaert M. G. (1986) Neither morphine nor clonidine influence the non-adrenergic, non-cholinergic inhibitory response in the rat gastric fundus. J. Pharm. Pharmaeol. 38, 621~522.
Biphasic response of rat gastric fundus Lefebvre R. A. (1986) Study on the possible neurotransmitter of the non-adrenergic, non-cholinergic innervation of the rat gastric fundus. Arch. Int. Pharmacodyn. 280, 110. Li. C. G. and Rand M. J. (1990) Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, noncholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur. J. Pharrnac. 4, 191 303-309. MacDonald A., Kelly J. and Dettmar P. W. (1990) Pre- and post-junctional ~t-adrenoceptor mediated response in the rat gastric fundus in vitro. J. Pharrn. Pharmacol. 42, 752-757. Muir T. C. and Smart N. G. (1983) The effects of clonidine on the response to stimulation of nonadrenergic, non-cholinergic nerves in the guinea-pig urinary bladder in vitro. J. Pharm. Pharmacol. 35, 234--237.
955
Shimo Y. and Ishii T. (1978) Effects of morphine on non-adrenergic, inhibitory response of the guinea-pig tania-coli. J. Pharm. Pharmacol. 30, 596-570. Starke K. (1981) ~t-adrenoceptor subclassification. Rev. Physiol. Biochem. Pharmac. 88, 199-236. Szerb J. C. (1982) Correlation between acetylcholine release and neuronal activity in the guinea-pig ileum myenteric plexus; effect of morphine. Neuroscience 7, 327-340. Verplanken P. A., Lefebvre R. A. and Bogaert M. G. (1984) Pharmacological characterization of ct-adrenoceptor in the rat gastric fundus. J. Pharmac. Exp. Ther. 231, 404-410. Yau W. M. and Lingle P. F. (1983) Interaction of enkephaline and caerulein on guinea-pig small intestine. Am. J. Physiol. 244, G65-G70.