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Pharmacological properties of a new centrally acting muscle relaxant (NC-1200) in isolated muscle preparations
Gen. Pharmac. Vol. 19, No. 1, pp. 117-121, 1988
0306-3623/88 $3.00+0.00 Copyright © 1988 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
PHARMACOLOGICAL PROPERTIES OF A NEW CENTRALLY ACTING MUSCLE RELAXANT (NC-1200) IN ISOLATED MUSCLE PREPARATIONS I. TAKAYANAGI, F. KONNO, Y. SATO and H. UNO Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan [Tel: (0474)72-1141]
(Received 12 February 1987)
Abstract--l. Pharmacological properties of a new centrally acting muscle relaxant (NC-1200) were tested in isolated muscle preparations. 2. NC-1200 acted as a Ca-blocker in the guinea pig taenia caecum. The pA2-value was 5.67. 3. In the rabbit aorta, NC-1200 competed with serotonin at serotonin receptors and also shifted the concentration response curves of histamine and norepinephrine suggesting the possibility that NC-1200 interacted with histamine and norepinephrine receptors. The pA2-value of NC-i 200 against serotonin was 6.02. 4. There was no evidence that NC-1200 interacted with drug-receptors in the muscles except the rabbit aorta. 5. The present results are similar to the previous findings that the properties of serotonin, histamine and norepinephrine receptors in the rabbit aorta were different from those in other muscles.
INTRODUCTION Centrally acting muscle relaxants, in general, affect spinal polysynaptic reflexes. Some muscle relaxants, such as tolperisone, afloqualone and baclofen, depress b o t h the m o n o - and poly-synaptic reflex potentials (Davidoff and Sears, 1974; Oehiai and Ishii, 1982). A newly developed centrally acting muscle relaxant, (4S,5R)-4-(2-methylpropyl)-3-[3-(perhydroazepin- 1-yl)-propyl- 1,3-oxazolidin-2-one (NC-1200) was reported to reduce markedly and severity o f the rat decerebrate rigidity, hardly affect spinal reflex potentials and little act on the end-plate and muscle. Therefore, NC-1200 is t h o u g h t to act on the central nervous system in a m a n n e r different from that o f other central muscle relaxant (Masaki and Shinozaki, 1986). However, a m o d e o f action o f NC-1200 on muscles was not studied. MATERIALS AND METHODS Male guinea pigs (300-450 g) of Hartley strain were killed by a blow on the neck and the entire ileum, taenia caecum and trachea were isolated. Longitudinal strips of ileum were isolated by carefully slipping an ileal segment (approximately 10cm long) over a tapered glass rod (Siegel and Triggie, 1982). A piece (about 3 cm) of the longitudinal muscle was suspended in 20 ml organ bath filled with a physiological solution (NaCI 154, KC1 5.6, MgCI 2 2.1, CaCI 2 2.2, NaHCO 3 5.9 and glucose 2.8 raM) gassed with a mixture of 95% 02 and 5% CO2 and kept at 32°C. The response to an agordst was recorded isotonicaUy under a tension of 0.5 g. In some experiments two platinum electrodes (2 × 45 ram) were set intervals of about 7 mm in the organ bath. The isolated ileal preparation was placed between two electrodes and field stimulation was carried out by passing a rectangular pulse of 300#see, supramaximal voltage and a frequency of 0.1 Hz between the two 117
electrodes. Responses to electrical stimulation were recorded isometrically with an initial tension of 0.5 g. Tracheal preparation was made by the method of Takagi and Takayanagi (1958) and mounted in the similar organ bath as described. The responses to a drug were recorded isotonically under a tension of 1 g. A piece (3-4 cm) of taenia caecum was suspended in 20 ml organ bath filled with Ca-free and isotonic 80 mM KC1 physiological solution, whose composition was NaCI 79.6mM, KCI 80mM, MgC12 2.1 mM, NaHCO 3 5.9mM and glucose 2.8 mM) gassed with a mixture of 95% 02 and 5% CO2 and kept at 32°C. In order to obtain a cumulative concentration response curve of CaCI2, responses to increasing concentrations of CaCI: was recorded isotonically under a tension of 0.5 g. Male Wistar strain rats (280-400 g) were killed by a blow on the head. The fundus and vas deferens were isolated. The fundus preparation was made according to Vane (1957). The fundus preparation and a piece (2-3 cm) of vas deferens were suspended in the similar organ bath as described. A tension was 1 g for the fundus preparation and 0.5 g for the vas deferens. Female Wistar rats (290--300 g) treated with estradiol benzoate (0.2 mg/kg s.c.) at 24 and 48 hr before sacrifice, female Wistar rats (300-380 g) castrated at one week before sacrifice and also female Wistar rats in the 17 and 18 day pregnancy were used. They were killed by a blow on the head and pieces (about 3 cm) of uterus were isolated. They were suspended in the similar organ bath as discussed. Responses to a drug were recorded isotonically under a tension of 0.5 g. Male rabbits (2.0-3.5 kg) were killed by bleeding from the neck. About 5 cm of thoracic aorta were dissected out close to the arch and cut helically. The aortic strips were then stripped of its endothelial cells by mechanical rubbing. A piece (about 2 mm in width and 3 cm in length) of aortic preparation was mounted in the similar bath as described. Responses to a drug were recorded isotonically under a tension of 2 g. In some experiments Ca-free physiological solution was used to a test a Ca-movement. Pieces (about
118
I. TAKAYANAGIet al.
3 x 4mm) of iris dilators from rabbits weighing 2.5 to 3.5 kg were dissected from the iris of the enucleated eyeball and mounted in the similar bath as described isometrically under a tension of 50 mg. A piece (about 2-3 cm) of ileum isolated from male rabbits (2.0-3.5 kg) was suspended in 20 ml organ bath as described. Responses were recorded isometrically under a tension of 1.5 g. Female frog (Rana nigromaculata) was killed and rectus abdominis m. was isolated. A piece (2-3 era) of rectus abdominis m. was suspended in a physiological solution (NaCI 111.2, KCI 1.9, CaC12 1.1 and NaH2PO 4 1.7 mM) kept at a room temperature and gassed with a mixture of 95% 02 and 5% CO2. Responses to drug were recorded isotonically under a tension of 0.5 g. Sea mussels, Mytilus edulis L., collected from the east side of Tokyo bay were used. Muscle bundle (about 1 mm in a diameter and about 2-3 cm in length) dissected from the anterior byssus retractor muscle under a pair of forceps were suspended in 20 ml organ bath filled with artificial sea water (NaC1 456, KC1 11, CaCI2.2H20 11, MgCI2-6H20 48 and Tris-HC1 25 mM) bubbled with a mixture of 95% O2 and 5% CO 2 and kept at 32°C. Responses to drugs were recorded under a tension of 0.5 g. Precise procedures were carried according to Takayanagi et al. (1981). All preparations were allowed to equilibrate for at least 60 min before addition of any drugs. The preparations except the anterior byssus retractor muscle were repeatedly exposed to a submaximum concentrations of an agonist used in each preparation until constant responses were obtained and then experiments were started. When norepinephrine was used as an agonist, the physiological solution used contained propranolol (10 -6 M), desmethylimipramine (10 -7 M) and normetanephrine (10 -6 M) to block fl-adrenoceptors and neural and extraneural uptake (Minneman and Abel, 1984). In order to estimate antagonistic potency of a drug, the preparations were equilibrated with an antagonist for 5 min, after determination of control concentration-response curves of the agonist, competitive antagonistic activity was expressed as a pA2-value, a negative log of dissociation constant of an antagonist which were calculated according to the method of Arunlakshana and Schild (1959). Drugs used: NC-1200 was kindly supplied from Nippon Chemiphar Pharmaceutical Company Ltd (Japan). Other drugs were obtained commercially, all in powder form.
400 --
/o
_~
o
I -9
-8
-7
-6
-5
Acetytchotine ( tog M ) Fig. 1. Concentration-response
curves of acetylcholine in
the absence and presence of NC-1200 in the guinea pig ileum. Ordinate: contraction (%) and abscissa: log concentration (M) of acetylcholine. O: acetylcholine alone, C): with NC-1200 (10 -6 M), A: with NC- 1200 (10 -5 M) and [-7: with NC-1200 0 0 -4 M). Each value is presented as a mean with SE of 6 experiments. 1200 was 4.15 + 0.24 (a mean + SE of 4 experiments). Simialr results were obtained in inhibition o f BaCI 2 (3 x 10 -4 M)-induced contraction and of the twitch response to field stimulation (Fig. 5). The pICs0-values of NC-1200 estimated in these results were 4.47 + 0.03 (a mean + SE of 8 experiments) and 4.14 + 0.13 (a mean + SE of 6 experiments), respectively. Inhibition o f the twitch response to field stimulation by norepinephrine (3 x 10-s-10 -5 M) was not influenced by NC-1200 (10 -5 and 10-4M), though NC-1200 (10-4M) depressed the twitch response slightly (10-20% of the twitch). 2. Guinea pig taenia caecum A concentration-response curve of CaCI2 was parallelly shifted by NC-1200 (10 -6, 10 -5 and 10-4M) concentration-dependently, suggesting a competitive
RESULTS
'1oo
1. Guinea pig ileum The ileum of guinea pig was slightly contracted by NC-1200 (more than 3 x 10-SM). C o n c e n t r a t i o n response curves o f acetylcholine and histamine were not influenced by NC-1200 (up to 10 -5 M). NC-1200 slightly contracted the ileum and almost completely inhibited the maximum responses to acetylcholine and histamine (Figs 1 and 2). A contractile response to NC-1200 was not influenced by atropine (10 -6 M), tetrodotoxin (3 x 10 -7 M) and by 2 hr treatment with capsaicin (10 -5 M) but completely inhibited by diphenhydramine (10 -7 M) (Fig. 3). A response of the ileum to nicotine (10 -5 M), which produced about 90% of the maximum to nicotine, was depressed by NC-1200 (more than 10 -5 M) (Fig. 4). Negative log o f concentration (M) induced 50% inhibition (pICs0) was 4.29 + 0.44 (a mean + SE of 4 experiments). NC-1200 (more than 10 -5 M) also depressed a response to serotonin (3 x 107 M) which was 80-90% of the maximum. The pICs0-value o f N C -
v
~5o
I -9
-8
I
I
-7 -6 Histamine (tog M )
I
-5
Fig. 2. Concentration-response curves of histamine in the absence and presence of NC-1200 in the guinea pig ileum. Ordinate: contraction (%) and abscissa: log concentration (M) of histamine. O: histamine alone, ©: with NC-1200 (10-~M), A: with NC-1200 (10-SM). 17:NC-1200 (10 -4 M). Each value is presented as a mean with SE of 6 experiments.
A new centrally acting muscle relaxant
119
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o I
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:.:
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111
-3
NC-1200 (tog M) 0
Fig. 3. Effect of diphenhydramine on a concentrationresponse curve of NC-1200 in the guinea pig ileum• Ordinate: percent of the contractile response to histamine, 10-5 M and abscissa: log concentration (M) ofNC-1200. O: NC-1200 alone and ©: with diphenhydramine (10-TM). Each point is presented as a mean with SE of 6 experiments•
Relaxation o f histamine ( 1 0 - S M ) - i n d u c c d cont r a c t i o n by isoproterenol 1 0 - 9 - 1 0 - 7 M ) was n o t influenced by NC-1200 ( 1 0 - S M ) . But NC-1200 (10 -4 M) relaxed it a l m o s t completely (Fig. 7).
.
.
.
.
A
B
C
Fig. 5. Effect of NC-1200 on the twitch response of the guinea pig ileum to field stimulation. Ordinate: twitch contraction (%). A: with NC-1200 (10-6M), B: with NC1200 (10-5 M) and C: with NC-1200 (10-4M). Each value is presented as a mean with SE of 6 experiments. *Significant difference from the control value at P < 0.05.
a n t a g o n i s m (Fig. 6). A Schild plot o f these results gave a straight line with a slope o f unity. The pA2-value o f NC-1200 against CaC12 was 5.67 _+ 0.19 (a m e a n + SE o f 6 experiments).
3. Guinea pig trachea
"'"
4oo
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-
/
0
4. Rat vas deferens Contractile responses to norepinephrine (10-7-10 -5 M) were n o t influenced by NC-1200 (up to 1 0 - S M ) b u t the m a x i m u m response to norepinephrine was depressed noncompetitively by N C 1200 (10 -4 M).
5. Rat fundus A c o n c e n t r a t i o n - r e s p o n s e curves o f serotonin was n o t influenced by 10 -5 a n d 10 -6 M o f NC-1200 b u t inhibited non-competitively by 10 -4 M o f NC-1200.
0
,~ -4
-5
of CaCl 2 in the guinea pig taenia caecum. Ordinate: contraction (%) and abscissa: log concentration (M) of CaCI 2. O: CaC12 alone, O: with NC-1200 (10-6M), A: with NC-1200 (10 -5 M), ["l: with NC-1200 (10 -4 M). Each point is presented as a mean with SE of 6 experiments.
A
-10 o
,//
I -1
CoLciurn chloride ( tog M )
:.:.: %.., %.., °.-., ....,
I -2
Fig. 6. Effect of NC-1200 on a concentration-response curve
L lOO
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Isoproterenot ( log M ) -9 -8 ,
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Fig. 4. Effect of NC-1200 on the contractile response of guinea pig ileum to nicotine. Ordinate: contraction (%). A: nicotine (10 -5 M) alone, B: with NC-1200 (]0 -7 M), C: with NC-1200 (10-6M), D: with NC-1200 (10-SM). E: with NC-1200 (10 -4 M) Each value is presented as a mean with SE of 4 experiments. *Significant difference from the value in A at P <0.05.
1oo Fig. 7. Concentration-response curves of isoproterenol on the guinea pig trachea contracted by histamine (10 -5 M) in the air,once and presence of NC-1200. Ordinate: relaxation (%) and abscissa: log concentration (M) of isoproterenol. O: isoproterenol alone, Z~: with NC-1200 (10 -5 M) and I~:
with NC-1200 (10 -4 M~. Each point is presented as a mean with SE of 4 expodments.
120
I. TAKAYANAGIet al.
100 :!:!: m 1 ::;21 :.:.: 5:': ,..%
r 7~
.% ,....
r----50
o -7
-6
-4 Histamine (Log M ) -5
-3
Fig. 8. Effect of NC-1200 on a concentration-response curves of histamine in the rabbit aorta. Ordinate: contraction (%) and abscissa: log concentration (M) of histamine. O: histamine alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10-SM) and I-1: with NC-1200 (10-4M). Each point is presented as a mean with SE of 4 experiments.
0
::::: :!:!:
y/,
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~'/
A
B
r-----
C
Fig. 10. Effect of NC-1200 on norepinephrine-induced contraction of the rabbit aorta in Ca-free physiological solution. Ordinate: contraction (%). A: norepinephrine (10-SM) alone, B: with NC-1200 (10-SM) and C: with NC-1200 (10 -4 M). Each value is presented as a mean with SE of 8 experiments. 7. Rabbit ileum
6. Rabbit thoracic aorta
A concentration-response curve of norepinephrine was shifted by 1 0 - 4 M of NC-1200 but not by 10 -5 and 1 0 - 6 M of NC-1200. Similar results were obtained in a antagonism between histamine and N C 1200 (Fig. 8). NC-1200 (10 -6, 10 -5 and 10-4M) shifted a concentration-response curve of serotonin in a concentration dependent manner (Fig. 9). A Schild plot of these results gave a straight line with a slope of unity, suggesting a competitive antagonism. The pA2-value was estimated as 6.02 + 0.21 (a mean _ SE of 6 experiments). After 6 0 m i n equilibrium in the normal physiological solution, the aortic strips were suspended in a Ca-free physiological solution containing NC-1200 (10 -4 M) for 5 min under these conditions, a response to norepinephrine (10-SM) was not influenced by NC-1200 (10 -4 M) (Fig. 10). Contractile response to prostaglandin F2= (10-7-10 -5 M) were not influenced by NC-1200 (10 -5 and 10 -4 M).
Spontaneous movement was inhibited by NC-1200 (10 -4 M). 8. Rabbit iris dilator
A concentration-response curve of norepinephrine was not influenced by 10 -6 and 10 -5 M of NC-1200 but inhibited non-competitively by its 10 -4 M. 9. R a t uterus
Contraction of uterus from the estradiol-treated rat induced by isotonic 30 m M KC1 physiological solution was relaxed by histamine 0 0 - 5 - 3 x 10 -3 M). NC-1200 (up to 10-4M) did not influence the concentration-response curve of histamine. In the uterus from the treated with estradiol, a concentration-response curve of serotonin was shifted by NC-1200 (10 -s M) with a decline of the maximum response and inhibited completely by 1 0 - 4 M of NC-1200 (Fig. 11). An antagonism between oxytocin and NC-1200 in the uterus from loo
100 --
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°
~ 5o
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5o
8 O
g
0 o
t"-8
-7
-6
I
-5
I
-4
Serotonin (log M ) Fig, 9. Effects of NC-1200 on a concentration-response curves of serotonin in the rabbit aorta. Ordinate: con-
traction (%) and abscissa: log concentration (M) of serotonin. O: serotonin alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10-SM) and I-q: with NC-1200 (10-4M). Each point is presented as a mean with SE of 6 experiments.
'~e/ -9
-8
-7
-6
-5
-4
Serotonin (tog M )
Fig. I1. Effect of NC-1200 on a concentration-response curve of serotonin in the uterus from the rat treated with estradiol. Ordinate: contraction (%) and abscissa: log concentration (M) of serotonin. 0 : serotonin alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10 -s M) and I--1: with NC-1200 (10 -4 M). Each point is presented as a mean with SE of 4 experiments.
121
A new centrally acting muscle relaxant castrated rat was almost same as mentioned above. In the uterus from the pregnant rat a contractile response to oxytocin (10 -3 unit/ml) was inhibited by 10-4M of NC-1200 but not by 10 -6 and 10 -SM. 10. Frog muscle (Rectus abdomines m.)
A concentration-response curve of acetylcholine was antagonized non-competitively by NC-1200 (10 6 and 10-SM). Further, NC-1200 in 10-4M completely inhibited by contractile responses to acetylcholine. 11. Molluscan smooth muscle (Anterior byssus retractor muscle)
Catch
contraction
induced
by
acetylcholine
(10 -4 M) was relaxed by NC-1200 (10-9-10 -5 M) in
a concentration-dependent manner. The relaxation by NC-1200 was not influenced by 3 x 10-5 M of haloperidol, which was a sufficient concentration to inhibit responses to dopamine (Yoshida et al. 1981).
DISCUSSION In the guinea pig taenia caecum, NC-1200 shifted the concentration-response curve of CaCI2 in a parallel fashion and the Schild plot for the antagonism between CaCI 2 and NC-1200 yielded a straight line with a slope of unity suggesting the competitive antagonism. Therefore NC-1200 is considered to be a Ca-blocker. Van Breemen et al. (1972) reported that norepinephrine-induced contraction of vascular smooth muscles was attributed to the facilitation of release of cellularly sequestered Ca. In Ca-freephysiological solution, contraction of rabbit aorta induced by norepinephrine, which was thought to be due to the release of Ca 2+ from the stores, was not influenced by NC-1200 (10-4M). These results (Fig. 10) suggested that NC-1200 did not inhibit Ca-release from the intracellular Ca-stores. Therefore, NC-1200 inhibits only Ca-influxes across the cell membranes. NC-1200 in higher concentrations more than 10-SM non-competitively antagonized the concentration-response curves of some agonists. These inhibitory actions of NC-1200 may be due to its Ca-blocking action. The present results indicated that NC-1200 did not interact with drug-receptors, acetylcholine (muscarinic and nicotinic), histamine, serotonin (D and M), ~t and fl-adrenaline, dopamine and oxytocin receptors in the muscles except the rabbit aorta. However, the antagonism between NC-1200 and serotonin in the rabbit aorta was thought to be competitive as the slope value from Schild analyses was not different from unity. The concentrationresponse curves of histamine and norepinephrine in the rabbit aorta were also parallelly shifted by NC1200 (10-4M) suggesting the possibility that the antagonisms between NC-1200 and histamine or norepinephrine were competitive. It is difficult to explain the phenomena that NC-1200 interacts with
the drug-receptors in the aortic muscle but not with those in other muscles. Ca-blockers, diltiazem and D600, were recently reported to compete with serotonin, histamine and norepinepbrine at their receptors in only rabbit aorta (Ohashi et al., 1985; 1986). These workers considered that pharmacological properties of these receptors in the rabbit aorta were different from those in other muscles. The present results confirm the view mentioned above. NC-1200 contracted the guinea pig ileum slightly. The contractile response of the ileum to NC-1200 was blocked by diphenhydramine, a histamine antagonist, but not by other antagonists. As the concentrationresponse curve of histamine was inhibited noncompetitively by NC-1200 the contractile response to NCoI200 was considered to be due to a histamine release. REFERENCES
Arunlakshana O. and Schild H. O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmac. 14, 48-58. Davidoff R. A. and Sears E. S. The effects of Lioresal on synaptic activity in the isolated spinal cord. Neurology 13, 957-963. Masaki M. and Shinozaki H. (1986) A new class of potent centrally acting muscle relaxants: pharmacology of oxazolidines in rat decerebrate rigidity. Br. J. Pharmac. 89, 2 i 9-228. Minneman K. P. and Abel P. W. (1984) Relationship between a~-adrenoceptor density and functional response of rat vas deferens. Studies with phenoxybenzamine. Naunyn-Schmiedebergs Arch Pharmac. 327, 238-246. Ochiai T. and Ishida R. (1982) Pharmacological studies on 6-amino-2-fluoromethyl- 3-(O-tolyl)-4-(3H)-quinazoline (afloqualone), a new centrally acting muscle relaxant. (II) Effects on the spinal reflex potential and the rigidity. Jap. J. Pharmac. 32, 427-438. Ohashi M., Kanai R. and Takayanagi I. (1985) Do D600 and diltiazem interact with serotonin receptors in rabbit vascular tissue? J. Pharmac. exp. Ther. 233, 830-835. Ohashi M., Takayanagi, I., Kumagai N., Iwata T. and Kuno E. (1986) Effects of Ca-blockers on contraction of an isolated rabbit vascular preparation induced by norepinephrine and histamine. Folia Pharmac. Japon. 88, 179P (in Japanese). Siegel H. and Triggle D. J. (1982) Benzilylcholinemustard and spare receptors in guinea pig ileum. Life Sci 30, 1645-1652. Takagi K. and Takayanagi I. (1958) Chemicopharmacological studies on antispasmodic action XV. Non-specific antispasmodic action on tracheal muscle. Chem. Pharm. Bull. 11, 379-383. Takayanagi I., Murakami H., Iwayama Y., Yoshida Y. and Miki S. (1981) Dopamine receptor in anterior byssus retractor muscle of mytilus edulis. Jap. J. Pharmac. 31, 249-252. Vane J. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. 12, 344-349. Van Breemen C., Farinas B. R., Greba P. and Naughton E. G. (1972) Excitation-contraction coupling in rabbit aorta studied by the Lanthanum method for measuring cellular calcium influx. Circulation Res. 30, 44-54. Yoshida Y., Takayanagi I. and Murakami H. (1981) Dopamine and its antagonists on molluscan smooth muscle. J. Pharmacobio-Dyn. 4, 226-228.
0306-3623/88 $3.00+0.00 Copyright © 1988 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
PHARMACOLOGICAL PROPERTIES OF A NEW CENTRALLY ACTING MUSCLE RELAXANT (NC-1200) IN ISOLATED MUSCLE PREPARATIONS I. TAKAYANAGI, F. KONNO, Y. SATO and H. UNO Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan [Tel: (0474)72-1141]
(Received 12 February 1987)
Abstract--l. Pharmacological properties of a new centrally acting muscle relaxant (NC-1200) were tested in isolated muscle preparations. 2. NC-1200 acted as a Ca-blocker in the guinea pig taenia caecum. The pA2-value was 5.67. 3. In the rabbit aorta, NC-1200 competed with serotonin at serotonin receptors and also shifted the concentration response curves of histamine and norepinephrine suggesting the possibility that NC-1200 interacted with histamine and norepinephrine receptors. The pA2-value of NC-i 200 against serotonin was 6.02. 4. There was no evidence that NC-1200 interacted with drug-receptors in the muscles except the rabbit aorta. 5. The present results are similar to the previous findings that the properties of serotonin, histamine and norepinephrine receptors in the rabbit aorta were different from those in other muscles.
INTRODUCTION Centrally acting muscle relaxants, in general, affect spinal polysynaptic reflexes. Some muscle relaxants, such as tolperisone, afloqualone and baclofen, depress b o t h the m o n o - and poly-synaptic reflex potentials (Davidoff and Sears, 1974; Oehiai and Ishii, 1982). A newly developed centrally acting muscle relaxant, (4S,5R)-4-(2-methylpropyl)-3-[3-(perhydroazepin- 1-yl)-propyl- 1,3-oxazolidin-2-one (NC-1200) was reported to reduce markedly and severity o f the rat decerebrate rigidity, hardly affect spinal reflex potentials and little act on the end-plate and muscle. Therefore, NC-1200 is t h o u g h t to act on the central nervous system in a m a n n e r different from that o f other central muscle relaxant (Masaki and Shinozaki, 1986). However, a m o d e o f action o f NC-1200 on muscles was not studied. MATERIALS AND METHODS Male guinea pigs (300-450 g) of Hartley strain were killed by a blow on the neck and the entire ileum, taenia caecum and trachea were isolated. Longitudinal strips of ileum were isolated by carefully slipping an ileal segment (approximately 10cm long) over a tapered glass rod (Siegel and Triggie, 1982). A piece (about 3 cm) of the longitudinal muscle was suspended in 20 ml organ bath filled with a physiological solution (NaCI 154, KC1 5.6, MgCI 2 2.1, CaCI 2 2.2, NaHCO 3 5.9 and glucose 2.8 raM) gassed with a mixture of 95% 02 and 5% CO2 and kept at 32°C. The response to an agordst was recorded isotonicaUy under a tension of 0.5 g. In some experiments two platinum electrodes (2 × 45 ram) were set intervals of about 7 mm in the organ bath. The isolated ileal preparation was placed between two electrodes and field stimulation was carried out by passing a rectangular pulse of 300#see, supramaximal voltage and a frequency of 0.1 Hz between the two 117
electrodes. Responses to electrical stimulation were recorded isometrically with an initial tension of 0.5 g. Tracheal preparation was made by the method of Takagi and Takayanagi (1958) and mounted in the similar organ bath as described. The responses to a drug were recorded isotonically under a tension of 1 g. A piece (3-4 cm) of taenia caecum was suspended in 20 ml organ bath filled with Ca-free and isotonic 80 mM KC1 physiological solution, whose composition was NaCI 79.6mM, KCI 80mM, MgC12 2.1 mM, NaHCO 3 5.9mM and glucose 2.8 mM) gassed with a mixture of 95% 02 and 5% CO2 and kept at 32°C. In order to obtain a cumulative concentration response curve of CaCI2, responses to increasing concentrations of CaCI: was recorded isotonically under a tension of 0.5 g. Male Wistar strain rats (280-400 g) were killed by a blow on the head. The fundus and vas deferens were isolated. The fundus preparation was made according to Vane (1957). The fundus preparation and a piece (2-3 cm) of vas deferens were suspended in the similar organ bath as described. A tension was 1 g for the fundus preparation and 0.5 g for the vas deferens. Female Wistar rats (290--300 g) treated with estradiol benzoate (0.2 mg/kg s.c.) at 24 and 48 hr before sacrifice, female Wistar rats (300-380 g) castrated at one week before sacrifice and also female Wistar rats in the 17 and 18 day pregnancy were used. They were killed by a blow on the head and pieces (about 3 cm) of uterus were isolated. They were suspended in the similar organ bath as discussed. Responses to a drug were recorded isotonically under a tension of 0.5 g. Male rabbits (2.0-3.5 kg) were killed by bleeding from the neck. About 5 cm of thoracic aorta were dissected out close to the arch and cut helically. The aortic strips were then stripped of its endothelial cells by mechanical rubbing. A piece (about 2 mm in width and 3 cm in length) of aortic preparation was mounted in the similar bath as described. Responses to a drug were recorded isotonically under a tension of 2 g. In some experiments Ca-free physiological solution was used to a test a Ca-movement. Pieces (about
118
I. TAKAYANAGIet al.
3 x 4mm) of iris dilators from rabbits weighing 2.5 to 3.5 kg were dissected from the iris of the enucleated eyeball and mounted in the similar bath as described isometrically under a tension of 50 mg. A piece (about 2-3 cm) of ileum isolated from male rabbits (2.0-3.5 kg) was suspended in 20 ml organ bath as described. Responses were recorded isometrically under a tension of 1.5 g. Female frog (Rana nigromaculata) was killed and rectus abdominis m. was isolated. A piece (2-3 era) of rectus abdominis m. was suspended in a physiological solution (NaCI 111.2, KCI 1.9, CaC12 1.1 and NaH2PO 4 1.7 mM) kept at a room temperature and gassed with a mixture of 95% 02 and 5% CO2. Responses to drug were recorded isotonically under a tension of 0.5 g. Sea mussels, Mytilus edulis L., collected from the east side of Tokyo bay were used. Muscle bundle (about 1 mm in a diameter and about 2-3 cm in length) dissected from the anterior byssus retractor muscle under a pair of forceps were suspended in 20 ml organ bath filled with artificial sea water (NaC1 456, KC1 11, CaCI2.2H20 11, MgCI2-6H20 48 and Tris-HC1 25 mM) bubbled with a mixture of 95% O2 and 5% CO 2 and kept at 32°C. Responses to drugs were recorded under a tension of 0.5 g. Precise procedures were carried according to Takayanagi et al. (1981). All preparations were allowed to equilibrate for at least 60 min before addition of any drugs. The preparations except the anterior byssus retractor muscle were repeatedly exposed to a submaximum concentrations of an agonist used in each preparation until constant responses were obtained and then experiments were started. When norepinephrine was used as an agonist, the physiological solution used contained propranolol (10 -6 M), desmethylimipramine (10 -7 M) and normetanephrine (10 -6 M) to block fl-adrenoceptors and neural and extraneural uptake (Minneman and Abel, 1984). In order to estimate antagonistic potency of a drug, the preparations were equilibrated with an antagonist for 5 min, after determination of control concentration-response curves of the agonist, competitive antagonistic activity was expressed as a pA2-value, a negative log of dissociation constant of an antagonist which were calculated according to the method of Arunlakshana and Schild (1959). Drugs used: NC-1200 was kindly supplied from Nippon Chemiphar Pharmaceutical Company Ltd (Japan). Other drugs were obtained commercially, all in powder form.
400 --
/o
_~
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Acetytchotine ( tog M ) Fig. 1. Concentration-response
curves of acetylcholine in
the absence and presence of NC-1200 in the guinea pig ileum. Ordinate: contraction (%) and abscissa: log concentration (M) of acetylcholine. O: acetylcholine alone, C): with NC-1200 (10 -6 M), A: with NC- 1200 (10 -5 M) and [-7: with NC-1200 0 0 -4 M). Each value is presented as a mean with SE of 6 experiments. 1200 was 4.15 + 0.24 (a mean + SE of 4 experiments). Simialr results were obtained in inhibition o f BaCI 2 (3 x 10 -4 M)-induced contraction and of the twitch response to field stimulation (Fig. 5). The pICs0-values of NC-1200 estimated in these results were 4.47 + 0.03 (a mean + SE of 8 experiments) and 4.14 + 0.13 (a mean + SE of 6 experiments), respectively. Inhibition o f the twitch response to field stimulation by norepinephrine (3 x 10-s-10 -5 M) was not influenced by NC-1200 (10 -5 and 10-4M), though NC-1200 (10-4M) depressed the twitch response slightly (10-20% of the twitch). 2. Guinea pig taenia caecum A concentration-response curve of CaCI2 was parallelly shifted by NC-1200 (10 -6, 10 -5 and 10-4M) concentration-dependently, suggesting a competitive
RESULTS
'1oo
1. Guinea pig ileum The ileum of guinea pig was slightly contracted by NC-1200 (more than 3 x 10-SM). C o n c e n t r a t i o n response curves o f acetylcholine and histamine were not influenced by NC-1200 (up to 10 -5 M). NC-1200 slightly contracted the ileum and almost completely inhibited the maximum responses to acetylcholine and histamine (Figs 1 and 2). A contractile response to NC-1200 was not influenced by atropine (10 -6 M), tetrodotoxin (3 x 10 -7 M) and by 2 hr treatment with capsaicin (10 -5 M) but completely inhibited by diphenhydramine (10 -7 M) (Fig. 3). A response of the ileum to nicotine (10 -5 M), which produced about 90% of the maximum to nicotine, was depressed by NC-1200 (more than 10 -5 M) (Fig. 4). Negative log o f concentration (M) induced 50% inhibition (pICs0) was 4.29 + 0.44 (a mean + SE of 4 experiments). NC-1200 (more than 10 -5 M) also depressed a response to serotonin (3 x 107 M) which was 80-90% of the maximum. The pICs0-value o f N C -
v
~5o
I -9
-8
I
I
-7 -6 Histamine (tog M )
I
-5
Fig. 2. Concentration-response curves of histamine in the absence and presence of NC-1200 in the guinea pig ileum. Ordinate: contraction (%) and abscissa: log concentration (M) of histamine. O: histamine alone, ©: with NC-1200 (10-~M), A: with NC-1200 (10-SM). 17:NC-1200 (10 -4 M). Each value is presented as a mean with SE of 6 experiments.
A new centrally acting muscle relaxant
119
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NC-1200 (tog M) 0
Fig. 3. Effect of diphenhydramine on a concentrationresponse curve of NC-1200 in the guinea pig ileum• Ordinate: percent of the contractile response to histamine, 10-5 M and abscissa: log concentration (M) ofNC-1200. O: NC-1200 alone and ©: with diphenhydramine (10-TM). Each point is presented as a mean with SE of 6 experiments•
Relaxation o f histamine ( 1 0 - S M ) - i n d u c c d cont r a c t i o n by isoproterenol 1 0 - 9 - 1 0 - 7 M ) was n o t influenced by NC-1200 ( 1 0 - S M ) . But NC-1200 (10 -4 M) relaxed it a l m o s t completely (Fig. 7).
.
.
.
.
A
B
C
Fig. 5. Effect of NC-1200 on the twitch response of the guinea pig ileum to field stimulation. Ordinate: twitch contraction (%). A: with NC-1200 (10-6M), B: with NC1200 (10-5 M) and C: with NC-1200 (10-4M). Each value is presented as a mean with SE of 6 experiments. *Significant difference from the control value at P < 0.05.
a n t a g o n i s m (Fig. 6). A Schild plot o f these results gave a straight line with a slope o f unity. The pA2-value o f NC-1200 against CaC12 was 5.67 _+ 0.19 (a m e a n + SE o f 6 experiments).
3. Guinea pig trachea
"'"
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/
0
4. Rat vas deferens Contractile responses to norepinephrine (10-7-10 -5 M) were n o t influenced by NC-1200 (up to 1 0 - S M ) b u t the m a x i m u m response to norepinephrine was depressed noncompetitively by N C 1200 (10 -4 M).
5. Rat fundus A c o n c e n t r a t i o n - r e s p o n s e curves o f serotonin was n o t influenced by 10 -5 a n d 10 -6 M o f NC-1200 b u t inhibited non-competitively by 10 -4 M o f NC-1200.
0
,~ -4
-5
of CaCl 2 in the guinea pig taenia caecum. Ordinate: contraction (%) and abscissa: log concentration (M) of CaCI 2. O: CaC12 alone, O: with NC-1200 (10-6M), A: with NC-1200 (10 -5 M), ["l: with NC-1200 (10 -4 M). Each point is presented as a mean with SE of 6 experiments.
A
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CoLciurn chloride ( tog M )
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Fig. 6. Effect of NC-1200 on a concentration-response curve
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Isoproterenot ( log M ) -9 -8 ,
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Fig. 4. Effect of NC-1200 on the contractile response of guinea pig ileum to nicotine. Ordinate: contraction (%). A: nicotine (10 -5 M) alone, B: with NC-1200 (]0 -7 M), C: with NC-1200 (10-6M), D: with NC-1200 (10-SM). E: with NC-1200 (10 -4 M) Each value is presented as a mean with SE of 4 experiments. *Significant difference from the value in A at P <0.05.
1oo Fig. 7. Concentration-response curves of isoproterenol on the guinea pig trachea contracted by histamine (10 -5 M) in the air,once and presence of NC-1200. Ordinate: relaxation (%) and abscissa: log concentration (M) of isoproterenol. O: isoproterenol alone, Z~: with NC-1200 (10 -5 M) and I~:
with NC-1200 (10 -4 M~. Each point is presented as a mean with SE of 4 expodments.
120
I. TAKAYANAGIet al.
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r 7~
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o -7
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Fig. 8. Effect of NC-1200 on a concentration-response curves of histamine in the rabbit aorta. Ordinate: contraction (%) and abscissa: log concentration (M) of histamine. O: histamine alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10-SM) and I-1: with NC-1200 (10-4M). Each point is presented as a mean with SE of 4 experiments.
0
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Fig. 10. Effect of NC-1200 on norepinephrine-induced contraction of the rabbit aorta in Ca-free physiological solution. Ordinate: contraction (%). A: norepinephrine (10-SM) alone, B: with NC-1200 (10-SM) and C: with NC-1200 (10 -4 M). Each value is presented as a mean with SE of 8 experiments. 7. Rabbit ileum
6. Rabbit thoracic aorta
A concentration-response curve of norepinephrine was shifted by 1 0 - 4 M of NC-1200 but not by 10 -5 and 1 0 - 6 M of NC-1200. Similar results were obtained in a antagonism between histamine and N C 1200 (Fig. 8). NC-1200 (10 -6, 10 -5 and 10-4M) shifted a concentration-response curve of serotonin in a concentration dependent manner (Fig. 9). A Schild plot of these results gave a straight line with a slope of unity, suggesting a competitive antagonism. The pA2-value was estimated as 6.02 + 0.21 (a mean _ SE of 6 experiments). After 6 0 m i n equilibrium in the normal physiological solution, the aortic strips were suspended in a Ca-free physiological solution containing NC-1200 (10 -4 M) for 5 min under these conditions, a response to norepinephrine (10-SM) was not influenced by NC-1200 (10 -4 M) (Fig. 10). Contractile response to prostaglandin F2= (10-7-10 -5 M) were not influenced by NC-1200 (10 -5 and 10 -4 M).
Spontaneous movement was inhibited by NC-1200 (10 -4 M). 8. Rabbit iris dilator
A concentration-response curve of norepinephrine was not influenced by 10 -6 and 10 -5 M of NC-1200 but inhibited non-competitively by its 10 -4 M. 9. R a t uterus
Contraction of uterus from the estradiol-treated rat induced by isotonic 30 m M KC1 physiological solution was relaxed by histamine 0 0 - 5 - 3 x 10 -3 M). NC-1200 (up to 10-4M) did not influence the concentration-response curve of histamine. In the uterus from the treated with estradiol, a concentration-response curve of serotonin was shifted by NC-1200 (10 -s M) with a decline of the maximum response and inhibited completely by 1 0 - 4 M of NC-1200 (Fig. 11). An antagonism between oxytocin and NC-1200 in the uterus from loo
100 --
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Serotonin (log M ) Fig, 9. Effects of NC-1200 on a concentration-response curves of serotonin in the rabbit aorta. Ordinate: con-
traction (%) and abscissa: log concentration (M) of serotonin. O: serotonin alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10-SM) and I-q: with NC-1200 (10-4M). Each point is presented as a mean with SE of 6 experiments.
'~e/ -9
-8
-7
-6
-5
-4
Serotonin (tog M )
Fig. I1. Effect of NC-1200 on a concentration-response curve of serotonin in the uterus from the rat treated with estradiol. Ordinate: contraction (%) and abscissa: log concentration (M) of serotonin. 0 : serotonin alone, ©: with NC-1200 (10 -6 M), A: with NC-1200 (10 -s M) and I--1: with NC-1200 (10 -4 M). Each point is presented as a mean with SE of 4 experiments.
121
A new centrally acting muscle relaxant castrated rat was almost same as mentioned above. In the uterus from the pregnant rat a contractile response to oxytocin (10 -3 unit/ml) was inhibited by 10-4M of NC-1200 but not by 10 -6 and 10 -SM. 10. Frog muscle (Rectus abdomines m.)
A concentration-response curve of acetylcholine was antagonized non-competitively by NC-1200 (10 6 and 10-SM). Further, NC-1200 in 10-4M completely inhibited by contractile responses to acetylcholine. 11. Molluscan smooth muscle (Anterior byssus retractor muscle)
Catch
contraction
induced
by
acetylcholine
(10 -4 M) was relaxed by NC-1200 (10-9-10 -5 M) in
a concentration-dependent manner. The relaxation by NC-1200 was not influenced by 3 x 10-5 M of haloperidol, which was a sufficient concentration to inhibit responses to dopamine (Yoshida et al. 1981).
DISCUSSION In the guinea pig taenia caecum, NC-1200 shifted the concentration-response curve of CaCI2 in a parallel fashion and the Schild plot for the antagonism between CaCI 2 and NC-1200 yielded a straight line with a slope of unity suggesting the competitive antagonism. Therefore NC-1200 is considered to be a Ca-blocker. Van Breemen et al. (1972) reported that norepinephrine-induced contraction of vascular smooth muscles was attributed to the facilitation of release of cellularly sequestered Ca. In Ca-freephysiological solution, contraction of rabbit aorta induced by norepinephrine, which was thought to be due to the release of Ca 2+ from the stores, was not influenced by NC-1200 (10-4M). These results (Fig. 10) suggested that NC-1200 did not inhibit Ca-release from the intracellular Ca-stores. Therefore, NC-1200 inhibits only Ca-influxes across the cell membranes. NC-1200 in higher concentrations more than 10-SM non-competitively antagonized the concentration-response curves of some agonists. These inhibitory actions of NC-1200 may be due to its Ca-blocking action. The present results indicated that NC-1200 did not interact with drug-receptors, acetylcholine (muscarinic and nicotinic), histamine, serotonin (D and M), ~t and fl-adrenaline, dopamine and oxytocin receptors in the muscles except the rabbit aorta. However, the antagonism between NC-1200 and serotonin in the rabbit aorta was thought to be competitive as the slope value from Schild analyses was not different from unity. The concentrationresponse curves of histamine and norepinephrine in the rabbit aorta were also parallelly shifted by NC1200 (10-4M) suggesting the possibility that the antagonisms between NC-1200 and histamine or norepinephrine were competitive. It is difficult to explain the phenomena that NC-1200 interacts with
the drug-receptors in the aortic muscle but not with those in other muscles. Ca-blockers, diltiazem and D600, were recently reported to compete with serotonin, histamine and norepinepbrine at their receptors in only rabbit aorta (Ohashi et al., 1985; 1986). These workers considered that pharmacological properties of these receptors in the rabbit aorta were different from those in other muscles. The present results confirm the view mentioned above. NC-1200 contracted the guinea pig ileum slightly. The contractile response of the ileum to NC-1200 was blocked by diphenhydramine, a histamine antagonist, but not by other antagonists. As the concentrationresponse curve of histamine was inhibited noncompetitively by NC-1200 the contractile response to NCoI200 was considered to be due to a histamine release. REFERENCES
Arunlakshana O. and Schild H. O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmac. 14, 48-58. Davidoff R. A. and Sears E. S. The effects of Lioresal on synaptic activity in the isolated spinal cord. Neurology 13, 957-963. Masaki M. and Shinozaki H. (1986) A new class of potent centrally acting muscle relaxants: pharmacology of oxazolidines in rat decerebrate rigidity. Br. J. Pharmac. 89, 2 i 9-228. Minneman K. P. and Abel P. W. (1984) Relationship between a~-adrenoceptor density and functional response of rat vas deferens. Studies with phenoxybenzamine. Naunyn-Schmiedebergs Arch Pharmac. 327, 238-246. Ochiai T. and Ishida R. (1982) Pharmacological studies on 6-amino-2-fluoromethyl- 3-(O-tolyl)-4-(3H)-quinazoline (afloqualone), a new centrally acting muscle relaxant. (II) Effects on the spinal reflex potential and the rigidity. Jap. J. Pharmac. 32, 427-438. Ohashi M., Kanai R. and Takayanagi I. (1985) Do D600 and diltiazem interact with serotonin receptors in rabbit vascular tissue? J. Pharmac. exp. Ther. 233, 830-835. Ohashi M., Takayanagi, I., Kumagai N., Iwata T. and Kuno E. (1986) Effects of Ca-blockers on contraction of an isolated rabbit vascular preparation induced by norepinephrine and histamine. Folia Pharmac. Japon. 88, 179P (in Japanese). Siegel H. and Triggle D. J. (1982) Benzilylcholinemustard and spare receptors in guinea pig ileum. Life Sci 30, 1645-1652. Takagi K. and Takayanagi I. (1958) Chemicopharmacological studies on antispasmodic action XV. Non-specific antispasmodic action on tracheal muscle. Chem. Pharm. Bull. 11, 379-383. Takayanagi I., Murakami H., Iwayama Y., Yoshida Y. and Miki S. (1981) Dopamine receptor in anterior byssus retractor muscle of mytilus edulis. Jap. J. Pharmac. 31, 249-252. Vane J. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. 12, 344-349. Van Breemen C., Farinas B. R., Greba P. and Naughton E. G. (1972) Excitation-contraction coupling in rabbit aorta studied by the Lanthanum method for measuring cellular calcium influx. Circulation Res. 30, 44-54. Yoshida Y., Takayanagi I. and Murakami H. (1981) Dopamine and its antagonists on molluscan smooth muscle. J. Pharmacobio-Dyn. 4, 226-228.