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Effect of cyclic AMP on mesaconitine-induced analgesia in mice
European Journal of Pharmacology, 108 (1985) 19-23
19
Elsevier
E F F E C T O F CYCLIC A M P O N M E S A C O N I T I N E - I N D U C E D ANALGESIA I N M I C E * MITSUO MU1LAYAMA and HIROSHI H I K I N O **
Pharmaceutical Institute, Tohoku University, Aoba- yarna, Sendai, Japan Received 13 March 1984, revised MS received 25 July 1984, accepted 2 October 1984
M. MURAYAMA and H. HIKINO, Effect of cyclic AMP on mesaconitine-induced analgesia in mice, European J. Pharmacol. 108 (1985) 19-23. The effects of cyclic AMP, dibutyryl cyclic AMP, theophylline, isoproterenol and propranolol on mesaconitine (MA)-induced antinociception in mice were investigated employing the tail flick and acetic acid-induced writhing methods for the evaluation of antinociceptive activity. MA-induced antinociception was significantly potentiated by cyclic AMP and dibutyryl cyclic AMP. The phosphodiesterase inhibitor theophylline also significantly potentiated the MA-induced antinociception. Further, MA-induced antinociception was markedly increased by a fl-adrenoceptor agonist, isoproterenol, and reduced by a fl-adrenoceptor antagonist, propranolol. These results suggest that the antinociceptive action of MA is potentiated through cyclic AMP and stimulation of the central fl-adrenergic system. Analgesia
Antinociception
fl-Adrenoceptor
Cyclic AMP
Mesaconitine
1. Introduction
2. Materials and methods
Mesaconitine (MA), the main alkaloid contained in clinically used aconite roots, has a potent analgesic action in mice (Hikino et al., 1979). As part of our effort to elucidate the mechanism of the analgesic action of MA, we recently demonstrated that the analgesic action of MA concerned the stimulation of the catecholaminergic system in the central nervous system (Murayama and Hikino, 1984). Catecholamines are known to markedly enhance the cyclic A M P level through activation of adenylate cyclase. Therefore, in the present study, the role of cyclic A M P in the potentiation of MA-induced antinociception was investigated by means of the tail flick and acetic acid-induced writhing methods.
2.1. Animals
* Pharmaceutical studies on Aconitum roots-Part 20. This paper also forms part 76 in the series on the validity of Oriental medicine. ** To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Male mice of the Std :ddY strain (20-25 g) were used. All mice were maintained on food and water ad libitum and conditioned at 24-25°C on a 12 h light-dark cycle.
2.2. Measurement of antinociceptive activity Antinociceptive activity was measured in the tail flick method ( D ' A m o u r and Smith, 1941) and the acetic acid-induced writhing method (Koster et al., 1959). Briefly, in the tail flick method, the ray was focused on a blackened spot on the tip of the mouse tail. The time elapsed before the response, a typical twitch of the tail, was measured. Antinociceptive activity was described as a percentage relative to the response time in mice before drug treatment (threshold percentage). In the acetic acid-induced writhing method, the mice were injected i.p. with physiological saline solution containing 0.7% acetic acid (10 ml/kg). The number
20
of writhing movements in mice was counted for 10 min from 10 min after the injection of acetic acid. Antinociceptive activity was recorded as a percentage relative to the writhing number of control mice (writhing percentage). Intracerebroventricular (i.c.v.) administration of drugs was done according to the method of Haley and McCormick (1957). MA was administered subcutaneously (s.c.) at 10 ~ g / k g in the tail flick method or 20 ~ g / k g in the acetic acid-induced writhing method 30 rain before tests. Cyclic AMP and dibutyryl cyclic AMP were given i.c.v, to mice 0, 0.5, 1, 2, 6 or 24 h before the administration of MA. Theophylline was injected s.c. 4, 12 or 24 h before the administration of MA. Isoproterenol and propranolol were given i.c.v, simultaneously with the injection of MA.
2.3. Drugs MA was prepared from Aconitum roots in our laboratory (Sato et al., 1979). The other substances used were acetic acid (Wako Pure Chemical), cyclic AMP (Sigma), N6,O2-dibutyryl cyclic AMP (dibutyryl cyclic AMP, Sigma), isoproterenol hydrochloride (Nikken Chemical), propranolol hydrochloride (Sumitomo Chemical) and theophylline (Wako Pure Chemical).
these cyclic nucleotides in the tail flick method while potentiation lasted until 24 h after i.c.v. administration in the acetic acid-induced writhing method (tables 1 and 2).
3.2. Effect of theophylline on mesaconitine-induced antinociception in mice Theophyiline (s.c.) alone did not exhibit any influence on the heat-induced tail twitch and acetic acid-induced writhing. According to both assay methods, theophylline markedly potentiated the MA-induced antinociception (table 3).
3.3. Effects of isoproterenol and propranolol on mesaconitine-induced antinociception in mice Isoproterenol (i.c.v.) alone did not have any effect on the heat-induced tail twitch. The MA-induced antinociception was enhanced by isoproterenol dose dependently (fig. 1). Propranolol (i.c.v.) alone did not affect the heat-induced tail twitch and acetic acid-induced
250
2.4. Statistical analysis of data 200
The data are expressed as means _+S.E. Oneway analysis of variance was used to evaluate the results in all experiments. 150
3. Results
3.1. Effects of cyclic A M P and dibutyryl cyclic A MP on mesaconitine-induced antinociception in mice
100 1---41
Cyclic AMP or dibutyryl cyclic AMP alone had no effect on the heat-induced tail twitch and acetic acid-induced writhing in the mice. The most potent increase of MA-induced antinociception caused by cyclic AMP or dibutyryl cyclic AMP in both assay methods was found within 1 h of i.c.v, administration. There was no subsequent potentiation by
0
1
I
I
I
I
0.01
0.1
0.8
1
,'~
Dose of isoi}roteren~fl ¢/ ~ g / m o u s { , ,
i. {'. ~.
Fig. 1. Effect of i s o p r o t e r e n o l o n m e s a c o n i t i n e - i n d u c e d antin o c i c e p t i o n in the tail flick m e t h o d (n = 7). Values w e r e exp r e s s e d as in M e t h o d s . I s o p r o t e r e n o l in saline w a s a d m i n i s t e r e d i.c.v, s i m u l t a n e o u s l y with s.c. injection of M A (O, 10 ~ t g / k g ) or saline ( I ) . S i g n i f i c a n t l y d i f f e r e n t f r o m the g r o u p t r e a t e d with b o t h saline (i.c.v.) a n d M A (s.c.), ** P < 0.01.
103_+ 9 95+ 4 100_+ 7
150_+ 7 171_+ 6* 183_+10"
153_+ 9 162_+12 167_+17 108_+ 7 109_+ 9 101_+ 7
95+ 6 111+ 6 102+10 167+13 186+13" 188+13"
155_+12 175_+12 188_+16"
148+ 6
MA
106_+10 97-+10 95_+10
97+15 99+ 8 100+ 7
104_+ 7
Control
1
148+ 9 164+19 185+18"
156± 3 186±16" 183_+18"
141+ 9
MA
107_+5 106-+7 111_+7
115+5 9g+4 117+2
102±9
Control
2
147+14 140+10 144± 8
148+ 9 148+ 7 145+ 4
143+12
MA
102+4 93_+9 100_+3
91_+6 95_+4 89_+5
96+4
Control
6
151+19 149+10 167+ 8
132_+ 6 148+ 8 135_+19
146+ 9
MA
106+5 106_+7 106_+7
99+7 1(18+6 94-+7
1(/2_+2
Control
24 (h) MA"
140_+18 147_+11 151_+ 9
156+ 9 152+15 152+ 4
150+13
95-t- 4 87_+12 87_+ 4 116_+ 7 87-+17 114-t- 9
30_+ 6 19_+ 3 * 11-+ 4** 48-+ 5 33_+ 7 * 24_+ 9*
107_+8 108_+9 111_+9 AMP 89-+7 108_+9 101_+5
100+12
Control
0.5
51_+11
MA
100_+5
Control
Writhing percentage (%) ~ 0
27_+5* 12_+3"* 5-+4**
54_+9 39_+5 13_+5"
55-+9
MA
1
105_+7 89_+8 93_+9
87-+3 94_+6 97+9
100+3
Control
33_+ 8 29_+ 4* 20_+ 6**
50-+12 37_+ 5* 30_+12"
59+ 5
MA
2
101_+ 6 85_+ 7 108_+ 5
105-+10 103_+ 4 110_+ 7
100-+ 4
Control
40_+ 8 31_+12 30+ 8*
48-+ 5 41+ 3 35_+ 3*
52_+ 7
MA
6
99_+ 9 91+ 4 104+ 7
110_+10 90_+19 80_+13
100-+ 3
Control
48_+8 35-+9 34_+2*
55_+9 42-+9 33+8*
60+6
MA
85_+ 9 97_+13 103__+ 8
90_+13 94_+ 6 103+ 3
100-+ 3
Control
24 (h) b
57_+5 63-+3 41-+3"
54_+9 55_+3 43-+4
61_+8
MA ~
a Values are expressed as in Methods. b The administration time of cyclic nucleotides in saline (i.c.v.) before administration of saline alone (control) or MA in saline (treated). c Saline or MA (20/~g/kg) was administered s.c. 30 min before the test. The writhing number of the control group given saline (i.c.v. and s.c.) was 25 + 3. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
0 Cyclic AMP 0.3 1.0 3.0 Dibutyryl cyclic 0.3 1.0 3.0
Dose of cyclic nucleotide (~g/mouse)
Effect of cyclic AMP and dibutyryl cyclic AMP administered i.c.v, on mesaconitine-induced antinociception in mice (acetic acid-induced writhing method) (n = 8).
TABLE 2
a Values are expressed as in Methods. ~' The administration time of cyclic nucleotides in saline (i.c.v.t before administration of saline alone (control) or MA in saline (treated). ~ Saline or MA (10 p,g/kg) was administered s.c. 30 rain before test. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05.
0,3 1.0 3.0
93-+ 7 102_+10 94_+ 8 AMP
0.3 1.0 3.0 Dibutyryl cyclic
105_+ 4
Control
144_+ 6
MA
Control
97+ 4
0.5
0
cyclic nucleotide (~g/mouse)
0 Cyclic AMP
Threshold percentage (%) ~
Dose of
Effect of cyclic AMP and dibutyryl cyclic AMP administered i.c.v, on mesaconitine-induced antinociception in mice (tail flick method) tn = 8l.
TABLE 1
22 TABLE 3
Effect of theophylline on mesaconitine-induced antinociception in mice (n = 8). Dose of
4
theophylline
Control
12
MA
Control
24 (h) ~ MA
Control
MA b
(mg/kg) Tail flick method (threshold percentage ~) 0 96+- 6 134+- 5 100 9 0+ 3 150+- 4 *
91_+8 89-+6
145+- 9 140+- 7
96+_5 91+4
141+ 4 138+ 6
100-+5 103+4 92+5 87+3
43+ 8 31±14 34+ 9 29+ 6
Acetic acid-induced writhing method (writhing percentage ~') 0 10 30 100
100+- 8 92_+ 5 97+- 8 108+_10
64+- 5 31+10" 4 1 ± 5* 46-+ 8*
100+-4 92_+8 86+5 86_+8
59+ 7 34+10 23+- 4** 19+- 4**
a The administration time of theophyUine in saline (s.c.) before administration of saline alone (control) or MA in saline (treated). b Saline or MA (10 p ,g/kg in the tail flick method, 2 0 / z g / k g in the acetic acid-induced writhing method) was administered s.c. 30 min before test. c Values are expressed as in Methods. Significantly different from the group treated with both saline (s.c.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
TABLE 4
Effect of propranolol on mesaconitine-induced antinociception in mice (n = 8). Dose of
Writhing percentage a
propranolol (/~ g / m o u s e )
Control
MA h
Control
MA b
0 0.3 1 3
100+ 9 97+- 12 88+ 9 91+-12
59+ 9 62+- 12 79_+12 100_+ 6*
101_+5 109+-7 111+-9 103+-8
182_+7 142_+ 8 ** 141_+7"* 140_+9"*
Threshold percentage"
a Values are expressed as in Methods. b Saline or MA ( 1 0 / ~ g / k g in the tail flick method, 20 ~ g / k g in the acetic acid-induced writhing method) was administered s,c. 30 min before test. Propranolol was administered i.c.v, simultaneously with MA. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
writhing but decreased the antinociceptive potency of MA (table 4).
4. Discussion In the previous study we had found that the analgesic action of MA on the heat-induced tail twitch and acetic acid-induced writhing was most potent 0.5 h after s.c. administration (Murayama and Hikino, 1984). The effects of cyclic AMP, dibutyryl cyclic AMP, theophylline, isoproterenol and propranolol on the MA-induced antinociception were therefore examined at 0.5 h after MA administration. The antinociceptive action of MA was significantly potentiated by the administration of cyclic
AMP, dibutyryl cyclic AMP or theophylline (tables 1, 2 and 3). These results suggest that the MA-induced antinociception was potentiated by an increase of cyclic AMP of exogenous and endogenous origin in the central nervous system. Cyclic AMP and dibutyryl cyclic AMP potentiated MA-induced antinociception most strongly within 1 h of i.c.v, administration. However, the potentiating effect of cyclic AMP or dibutyryl cyclic AMP on the MA-induced decrease of writhing was observed even 24 h after the administration in the acetic acid-induced writhing method. This prolonged potentiating effect of cyclic AMP or dibutyryl cyclic AMP on the MA-induced reduction of writhing movements in the acetic acidinduced writhing method may have been related to the fact that acetic acid-induced writhing is subject
23
to effects other than antinociception, since the prolonged potentiating effects of these cyclic nucleotides in the acetic acid-induced writhing method were not observed in the tail flick method. Previously, we demonstrated that MA-induced analgesia was enhanced through the activation of the central catecholaminergic system and that MA promoted the turnover rate of noradrenaline in the central nervous system (Murayama and Hikino, 1984). Noradrenaline is known to activate adenylate cyclase through fl-adrenoceptors and markedly increase the level of cyclic AMP (Hazeki and Ui, 1980). Therefore, the effects of a fl-adrenoceptor agonist, isoproterenol, and a fl-adrenoceptor antagonist, propranolol, on the MA-induced antinociception were investigated. As shown in table 4 and fig. 1, the antinociceptive action of MA was significantly potentiated by isoproterenol while it was suppressed by propranolol. Accordingly, MA-induced antinociception appears to be affected through the activation of the central ~8adrenergic system. On the basis of the above evidence, it is sug-
gested that the antinociceptive action of MA is potentiated through cyclic AMP and stimulation of the central fl-adrenergic system.
References D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Hazeki, O. and M. Ui, 1980, Beta 1- and 2-adrenergic receptor responsible for cyclic AMP accumulation in isolated heart and lung cells, Mol. Pharmacol. 17, 8. Hikino, H., T. Ito, C. Konno and Y. Ohizumi, 1979, Analgesic principles of A c o n i t u m roots, J. Pharm. Dyn. 2, 78. Koster, R., M. Anderson and E.J. De Beer, 1959, Acetic acid for analgesic screening, Fed. Proc. 18, 412. Murayama, M. and H. Hikino, 1984, Mechanism of analgesic action of mesaconitine: Relationship between analgesic effect and central monoamines or opiate receptors, European J. Pharmacol. 101, 29. Soto, H., C. Yamada, C. Konno, Y. Ohizumi, K. Endo and H. Hikino, 1979, Pharmacological actions of aconitine alkaloids, Tohoku J. Exp. Med. 128, 175.
19
Elsevier
E F F E C T O F CYCLIC A M P O N M E S A C O N I T I N E - I N D U C E D ANALGESIA I N M I C E * MITSUO MU1LAYAMA and HIROSHI H I K I N O **
Pharmaceutical Institute, Tohoku University, Aoba- yarna, Sendai, Japan Received 13 March 1984, revised MS received 25 July 1984, accepted 2 October 1984
M. MURAYAMA and H. HIKINO, Effect of cyclic AMP on mesaconitine-induced analgesia in mice, European J. Pharmacol. 108 (1985) 19-23. The effects of cyclic AMP, dibutyryl cyclic AMP, theophylline, isoproterenol and propranolol on mesaconitine (MA)-induced antinociception in mice were investigated employing the tail flick and acetic acid-induced writhing methods for the evaluation of antinociceptive activity. MA-induced antinociception was significantly potentiated by cyclic AMP and dibutyryl cyclic AMP. The phosphodiesterase inhibitor theophylline also significantly potentiated the MA-induced antinociception. Further, MA-induced antinociception was markedly increased by a fl-adrenoceptor agonist, isoproterenol, and reduced by a fl-adrenoceptor antagonist, propranolol. These results suggest that the antinociceptive action of MA is potentiated through cyclic AMP and stimulation of the central fl-adrenergic system. Analgesia
Antinociception
fl-Adrenoceptor
Cyclic AMP
Mesaconitine
1. Introduction
2. Materials and methods
Mesaconitine (MA), the main alkaloid contained in clinically used aconite roots, has a potent analgesic action in mice (Hikino et al., 1979). As part of our effort to elucidate the mechanism of the analgesic action of MA, we recently demonstrated that the analgesic action of MA concerned the stimulation of the catecholaminergic system in the central nervous system (Murayama and Hikino, 1984). Catecholamines are known to markedly enhance the cyclic A M P level through activation of adenylate cyclase. Therefore, in the present study, the role of cyclic A M P in the potentiation of MA-induced antinociception was investigated by means of the tail flick and acetic acid-induced writhing methods.
2.1. Animals
* Pharmaceutical studies on Aconitum roots-Part 20. This paper also forms part 76 in the series on the validity of Oriental medicine. ** To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Male mice of the Std :ddY strain (20-25 g) were used. All mice were maintained on food and water ad libitum and conditioned at 24-25°C on a 12 h light-dark cycle.
2.2. Measurement of antinociceptive activity Antinociceptive activity was measured in the tail flick method ( D ' A m o u r and Smith, 1941) and the acetic acid-induced writhing method (Koster et al., 1959). Briefly, in the tail flick method, the ray was focused on a blackened spot on the tip of the mouse tail. The time elapsed before the response, a typical twitch of the tail, was measured. Antinociceptive activity was described as a percentage relative to the response time in mice before drug treatment (threshold percentage). In the acetic acid-induced writhing method, the mice were injected i.p. with physiological saline solution containing 0.7% acetic acid (10 ml/kg). The number
20
of writhing movements in mice was counted for 10 min from 10 min after the injection of acetic acid. Antinociceptive activity was recorded as a percentage relative to the writhing number of control mice (writhing percentage). Intracerebroventricular (i.c.v.) administration of drugs was done according to the method of Haley and McCormick (1957). MA was administered subcutaneously (s.c.) at 10 ~ g / k g in the tail flick method or 20 ~ g / k g in the acetic acid-induced writhing method 30 rain before tests. Cyclic AMP and dibutyryl cyclic AMP were given i.c.v, to mice 0, 0.5, 1, 2, 6 or 24 h before the administration of MA. Theophylline was injected s.c. 4, 12 or 24 h before the administration of MA. Isoproterenol and propranolol were given i.c.v, simultaneously with the injection of MA.
2.3. Drugs MA was prepared from Aconitum roots in our laboratory (Sato et al., 1979). The other substances used were acetic acid (Wako Pure Chemical), cyclic AMP (Sigma), N6,O2-dibutyryl cyclic AMP (dibutyryl cyclic AMP, Sigma), isoproterenol hydrochloride (Nikken Chemical), propranolol hydrochloride (Sumitomo Chemical) and theophylline (Wako Pure Chemical).
these cyclic nucleotides in the tail flick method while potentiation lasted until 24 h after i.c.v. administration in the acetic acid-induced writhing method (tables 1 and 2).
3.2. Effect of theophylline on mesaconitine-induced antinociception in mice Theophyiline (s.c.) alone did not exhibit any influence on the heat-induced tail twitch and acetic acid-induced writhing. According to both assay methods, theophylline markedly potentiated the MA-induced antinociception (table 3).
3.3. Effects of isoproterenol and propranolol on mesaconitine-induced antinociception in mice Isoproterenol (i.c.v.) alone did not have any effect on the heat-induced tail twitch. The MA-induced antinociception was enhanced by isoproterenol dose dependently (fig. 1). Propranolol (i.c.v.) alone did not affect the heat-induced tail twitch and acetic acid-induced
250
2.4. Statistical analysis of data 200
The data are expressed as means _+S.E. Oneway analysis of variance was used to evaluate the results in all experiments. 150
3. Results
3.1. Effects of cyclic A M P and dibutyryl cyclic A MP on mesaconitine-induced antinociception in mice
100 1---41
Cyclic AMP or dibutyryl cyclic AMP alone had no effect on the heat-induced tail twitch and acetic acid-induced writhing in the mice. The most potent increase of MA-induced antinociception caused by cyclic AMP or dibutyryl cyclic AMP in both assay methods was found within 1 h of i.c.v, administration. There was no subsequent potentiation by
0
1
I
I
I
I
0.01
0.1
0.8
1
,'~
Dose of isoi}roteren~fl ¢/ ~ g / m o u s { , ,
i. {'. ~.
Fig. 1. Effect of i s o p r o t e r e n o l o n m e s a c o n i t i n e - i n d u c e d antin o c i c e p t i o n in the tail flick m e t h o d (n = 7). Values w e r e exp r e s s e d as in M e t h o d s . I s o p r o t e r e n o l in saline w a s a d m i n i s t e r e d i.c.v, s i m u l t a n e o u s l y with s.c. injection of M A (O, 10 ~ t g / k g ) or saline ( I ) . S i g n i f i c a n t l y d i f f e r e n t f r o m the g r o u p t r e a t e d with b o t h saline (i.c.v.) a n d M A (s.c.), ** P < 0.01.
103_+ 9 95+ 4 100_+ 7
150_+ 7 171_+ 6* 183_+10"
153_+ 9 162_+12 167_+17 108_+ 7 109_+ 9 101_+ 7
95+ 6 111+ 6 102+10 167+13 186+13" 188+13"
155_+12 175_+12 188_+16"
148+ 6
MA
106_+10 97-+10 95_+10
97+15 99+ 8 100+ 7
104_+ 7
Control
1
148+ 9 164+19 185+18"
156± 3 186±16" 183_+18"
141+ 9
MA
107_+5 106-+7 111_+7
115+5 9g+4 117+2
102±9
Control
2
147+14 140+10 144± 8
148+ 9 148+ 7 145+ 4
143+12
MA
102+4 93_+9 100_+3
91_+6 95_+4 89_+5
96+4
Control
6
151+19 149+10 167+ 8
132_+ 6 148+ 8 135_+19
146+ 9
MA
106+5 106_+7 106_+7
99+7 1(18+6 94-+7
1(/2_+2
Control
24 (h) MA"
140_+18 147_+11 151_+ 9
156+ 9 152+15 152+ 4
150+13
95-t- 4 87_+12 87_+ 4 116_+ 7 87-+17 114-t- 9
30_+ 6 19_+ 3 * 11-+ 4** 48-+ 5 33_+ 7 * 24_+ 9*
107_+8 108_+9 111_+9 AMP 89-+7 108_+9 101_+5
100+12
Control
0.5
51_+11
MA
100_+5
Control
Writhing percentage (%) ~ 0
27_+5* 12_+3"* 5-+4**
54_+9 39_+5 13_+5"
55-+9
MA
1
105_+7 89_+8 93_+9
87-+3 94_+6 97+9
100+3
Control
33_+ 8 29_+ 4* 20_+ 6**
50-+12 37_+ 5* 30_+12"
59+ 5
MA
2
101_+ 6 85_+ 7 108_+ 5
105-+10 103_+ 4 110_+ 7
100-+ 4
Control
40_+ 8 31_+12 30+ 8*
48-+ 5 41+ 3 35_+ 3*
52_+ 7
MA
6
99_+ 9 91+ 4 104+ 7
110_+10 90_+19 80_+13
100-+ 3
Control
48_+8 35-+9 34_+2*
55_+9 42-+9 33+8*
60+6
MA
85_+ 9 97_+13 103__+ 8
90_+13 94_+ 6 103+ 3
100-+ 3
Control
24 (h) b
57_+5 63-+3 41-+3"
54_+9 55_+3 43-+4
61_+8
MA ~
a Values are expressed as in Methods. b The administration time of cyclic nucleotides in saline (i.c.v.) before administration of saline alone (control) or MA in saline (treated). c Saline or MA (20/~g/kg) was administered s.c. 30 min before the test. The writhing number of the control group given saline (i.c.v. and s.c.) was 25 + 3. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
0 Cyclic AMP 0.3 1.0 3.0 Dibutyryl cyclic 0.3 1.0 3.0
Dose of cyclic nucleotide (~g/mouse)
Effect of cyclic AMP and dibutyryl cyclic AMP administered i.c.v, on mesaconitine-induced antinociception in mice (acetic acid-induced writhing method) (n = 8).
TABLE 2
a Values are expressed as in Methods. ~' The administration time of cyclic nucleotides in saline (i.c.v.t before administration of saline alone (control) or MA in saline (treated). ~ Saline or MA (10 p,g/kg) was administered s.c. 30 rain before test. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05.
0,3 1.0 3.0
93-+ 7 102_+10 94_+ 8 AMP
0.3 1.0 3.0 Dibutyryl cyclic
105_+ 4
Control
144_+ 6
MA
Control
97+ 4
0.5
0
cyclic nucleotide (~g/mouse)
0 Cyclic AMP
Threshold percentage (%) ~
Dose of
Effect of cyclic AMP and dibutyryl cyclic AMP administered i.c.v, on mesaconitine-induced antinociception in mice (tail flick method) tn = 8l.
TABLE 1
22 TABLE 3
Effect of theophylline on mesaconitine-induced antinociception in mice (n = 8). Dose of
4
theophylline
Control
12
MA
Control
24 (h) ~ MA
Control
MA b
(mg/kg) Tail flick method (threshold percentage ~) 0 96+- 6 134+- 5 100 9 0+ 3 150+- 4 *
91_+8 89-+6
145+- 9 140+- 7
96+_5 91+4
141+ 4 138+ 6
100-+5 103+4 92+5 87+3
43+ 8 31±14 34+ 9 29+ 6
Acetic acid-induced writhing method (writhing percentage ~') 0 10 30 100
100+- 8 92_+ 5 97+- 8 108+_10
64+- 5 31+10" 4 1 ± 5* 46-+ 8*
100+-4 92_+8 86+5 86_+8
59+ 7 34+10 23+- 4** 19+- 4**
a The administration time of theophyUine in saline (s.c.) before administration of saline alone (control) or MA in saline (treated). b Saline or MA (10 p ,g/kg in the tail flick method, 2 0 / z g / k g in the acetic acid-induced writhing method) was administered s.c. 30 min before test. c Values are expressed as in Methods. Significantly different from the group treated with both saline (s.c.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
TABLE 4
Effect of propranolol on mesaconitine-induced antinociception in mice (n = 8). Dose of
Writhing percentage a
propranolol (/~ g / m o u s e )
Control
MA h
Control
MA b
0 0.3 1 3
100+ 9 97+- 12 88+ 9 91+-12
59+ 9 62+- 12 79_+12 100_+ 6*
101_+5 109+-7 111+-9 103+-8
182_+7 142_+ 8 ** 141_+7"* 140_+9"*
Threshold percentage"
a Values are expressed as in Methods. b Saline or MA ( 1 0 / ~ g / k g in the tail flick method, 20 ~ g / k g in the acetic acid-induced writhing method) was administered s,c. 30 min before test. Propranolol was administered i.c.v, simultaneously with MA. Significantly different from the group treated with both saline (i.c.v.) and MA (s.c.), * P < 0.05 or ** P < 0.01.
writhing but decreased the antinociceptive potency of MA (table 4).
4. Discussion In the previous study we had found that the analgesic action of MA on the heat-induced tail twitch and acetic acid-induced writhing was most potent 0.5 h after s.c. administration (Murayama and Hikino, 1984). The effects of cyclic AMP, dibutyryl cyclic AMP, theophylline, isoproterenol and propranolol on the MA-induced antinociception were therefore examined at 0.5 h after MA administration. The antinociceptive action of MA was significantly potentiated by the administration of cyclic
AMP, dibutyryl cyclic AMP or theophylline (tables 1, 2 and 3). These results suggest that the MA-induced antinociception was potentiated by an increase of cyclic AMP of exogenous and endogenous origin in the central nervous system. Cyclic AMP and dibutyryl cyclic AMP potentiated MA-induced antinociception most strongly within 1 h of i.c.v, administration. However, the potentiating effect of cyclic AMP or dibutyryl cyclic AMP on the MA-induced decrease of writhing was observed even 24 h after the administration in the acetic acid-induced writhing method. This prolonged potentiating effect of cyclic AMP or dibutyryl cyclic AMP on the MA-induced reduction of writhing movements in the acetic acidinduced writhing method may have been related to the fact that acetic acid-induced writhing is subject
23
to effects other than antinociception, since the prolonged potentiating effects of these cyclic nucleotides in the acetic acid-induced writhing method were not observed in the tail flick method. Previously, we demonstrated that MA-induced analgesia was enhanced through the activation of the central catecholaminergic system and that MA promoted the turnover rate of noradrenaline in the central nervous system (Murayama and Hikino, 1984). Noradrenaline is known to activate adenylate cyclase through fl-adrenoceptors and markedly increase the level of cyclic AMP (Hazeki and Ui, 1980). Therefore, the effects of a fl-adrenoceptor agonist, isoproterenol, and a fl-adrenoceptor antagonist, propranolol, on the MA-induced antinociception were investigated. As shown in table 4 and fig. 1, the antinociceptive action of MA was significantly potentiated by isoproterenol while it was suppressed by propranolol. Accordingly, MA-induced antinociception appears to be affected through the activation of the central ~8adrenergic system. On the basis of the above evidence, it is sug-
gested that the antinociceptive action of MA is potentiated through cyclic AMP and stimulation of the central fl-adrenergic system.
References D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Br. J. Pharmacol. 12, 12. Hazeki, O. and M. Ui, 1980, Beta 1- and 2-adrenergic receptor responsible for cyclic AMP accumulation in isolated heart and lung cells, Mol. Pharmacol. 17, 8. Hikino, H., T. Ito, C. Konno and Y. Ohizumi, 1979, Analgesic principles of A c o n i t u m roots, J. Pharm. Dyn. 2, 78. Koster, R., M. Anderson and E.J. De Beer, 1959, Acetic acid for analgesic screening, Fed. Proc. 18, 412. Murayama, M. and H. Hikino, 1984, Mechanism of analgesic action of mesaconitine: Relationship between analgesic effect and central monoamines or opiate receptors, European J. Pharmacol. 101, 29. Soto, H., C. Yamada, C. Konno, Y. Ohizumi, K. Endo and H. Hikino, 1979, Pharmacological actions of aconitine alkaloids, Tohoku J. Exp. Med. 128, 175.