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Intracerebroventricular administration of angiotensin II attenuates morphine-induced analgesia in mice
Neuropharmacology Vol.24,No. 11,pp. 1131-1134, 1985 Printed inGreatBritain. Allrights reserved
INTRACEREBROVENTRICULAR
ADMINISTRATION
0028-3908/85 $3.00+0.00 Copyright 0 1985PergamonPressLtd
OF ANGIOTENSIN
ANALGESIA S. Kaneko, Department
Faculty
Kyoto University,
IAccepXed
MORPHINE-INDUCED
IN MICE
A. Mori, S. Tamura,
of Pharmacology,
II ATTENUATES
M. Satoh and H. Takagi of Pharmaceutical
Sciences,
Kyoto 606, Japan.
23 Sepmnben.
1985)
Summary - The central effect of angiotensin II on the analgesic action of morphine in mice was investigated using the tail-pinch and hot plate tests. Angiotensin II (0.1-10 pmol), given intracerebroventricularly (i.c.v.), had no effect on the nociceptive sensitivity but did produce a dose-dependent attenuation of the morphine-induced analgesia. A specific angiotensin II antagonist, Saralasin (1 pmol), which in itself had no analgesic effect, significantly potentiated the morphine-induced analgesia. These results suggest that angiotensin II probably plays the physiological role of an anti-opioid substance in pain-modulating systems in the brain.
Endogenous opioid substances, extracted from bovine brain, have been purified and shown to be angiotensin I and II. It was also shown that angiotensin II antagonized the inhibitory effect of Met-enkephalin in the electrically-stimulated guinea-pig ileum preparation, in a physiological manner (Kaneko, Tamura and Takagi, 1985). Angiotensin II has several different pharmacological effects on the central nervous system (Ganten, Lang, Lehmann and Ungar, 1984) and some of these effects are inhibited by opioid peptides (Summy-long, Keil, Deen, Rosella and Severs, 1981). It has been proposed that angiotensins and enkephalins are cleaved by common enzymes in the brain. Angiotensin converting enzyme (EC 3.4.15.1) which converts angiotensin I to angiotensin II, also degrades enkephalin (Stine, Yang and Costa, 1980). Dipeptidyl-aminopeptidase III (EC 3.4.14.4) shows a highly selective affinity for angiotensin EI and enkephalins (Lee and Snyder, 1982). Moreover, earlier studies demonstrated that the output of acetylcholine (ACh 1 from the surface of the cerebral cortex in the cat was decreased by morphine (Jhamandas, Pinsky and Phillis,1970), while it was increased by angiotensin II (Elie and Panisset,lQ?O) However, the actual relationship between angiotensins and opioids in the regulation of pain in the brain is not well understood. brain, mice.
Based on the assumption that angiotensin II may have anti-opioid effects in the the effect of angiotensin II on analgesia induced by morphine was examined in ViVO in
METHODS II (saralasin) were [Ile5]-Angiotensin II and [Sar' ,Val',Ala']-angiotensin purchased from the Protein Research Foundation, Osaka, Japan. These peptides and morphine hydrochloride were dissolved in physiological saline just prior to use. Experiments were carried out on male dd-K mice (15-20 g). Each mouse was given a single intracerebroventricular injection of the drugs in a volume of 10 ~1, using the method of Haley and McCormick (1957). When examining the effect of angiotensin II or saralasin on morphine-induced analgesia, these compounds were mixed together and injected. Analgesia was evaluated by the tail-pinch test (Takagi, Inukai and Nakama, 1966) or the hot plate test. In the tail-pinch test, an aryery clip with a constant pressure of 200 g was used. Mice were used which bit the clip within 2 set after being pinched at the base of the tail, including the anal mucosa, one hour prior to injection. The criterion of analgesia, after the injection of drugs, was an inhibition of the biting response within 6 sec. The results were expressed as the percentage of mice which showed analgesia. In the hot plate test, the mice were placed on a hot stainless steel plate, maintained at 52 f l°C, and the patency of hindpaw-licking or jumping was measured. The baseline latency (To) was first obtained from the mean of two latency values observed 30 and 15 min prior to injection, then the test latency (T) was determined after the injection of drugs, where the cut-off time was 60 sec. The analgesic index (as a percentage [Xl) was calculated as:
?r.P 14
I ,-_I
1131
1132
Preliminary
(T-T0)/(60-To)xlOO.
Notes
The mean f SEM of the To was 21.8tO.4
set (number of animals;
n = 96).
While the analgesic tests were being done, the Straub's tail response, a characteristic excitatory behavioural response of mice, induced by morphine, was also scored. The Straub's tail response was graded by the angle of the tail top to horizontal as: 0°-450 = 0, 45O-90° = 1, more than 90° = 2, at 15, 30, 45, 60 and 90 min after the injection, and the Straub's tail score was determined as the mean of added values of the response per mouse. tail-pinch
Statistical significance was analyzed by Fishers's exact probability test and unpaired t-test (one-tailed) in the hot plate test.
test in the
RESULTS As shown in Figure lA, when angiotensin II (10 pmol) was injected, together with morphine (2.5 nmol)l the analgesic effect of morphine was attenuated for the entire timecourse of the tail-pinch teat, however, the Straub's taile score was not influenced by angiotensin II (group given morphine = 2.53fU.24; group given morphine + angiotensin II = 2.50f0.56). Angiotensin II, when injected alone, did not produced analgesia or abnormal behaviour. Figure 2 summarizes the extent of the suppressant effect of angiotensin II on the analgesia induced by morphine in the tail-pinch test. This effect was dose-dependent in the range 0.1-10 pm01 of angiotensin II. A maximum attenuation was obtained at 10 pmol, but larger doses of angiotensin II were less effective than 10 pmol. A much larger dose of angiotensin II (1000 pmol) had no anti-analgesic effect and no analgesic effect was observed when this dose of angiotensin II was injected alone. In the hot plate test, angiotensin II (10 pmol) alone produced no change in hindpaw-licking or jumping latencies, as compared with the group given saline, but the suppressant effect on morphine-induced analgesia was also observed when angiotensin II was injected together with morphine (Table 1).
Table
1. Effect
of angiotensin
II on morphine-induced
analgesia
in mice
(Hot plate test)
Intracerebroventricular
Minutes
(n)
administration
after injection
15
30
45
60
90
(IO)
-2f8
-4t8
-1f6
-5+8
-6f7
10 pm01
(10)
1+4
-1+6
-1f5
-8k8
-7+9
Morphine
2.5 nmol
(18)
78f8
6'/+9
62+9
43+9
19+4
Morphine + AGT II
2.5 nmol 10 pm01
(18)
48?r9* 45f9*
33t8*
3129
13t8
Saline AGT II
Values
are analgesic
AGT II = angiotensin
index (mean t SEM %).
* P < 0.05 vs. morphine
alone
II.
The effect of saralasin, a specific angiotensin II receptor antagonist, on morphine-induced analgesia was examined in order to determine whether endogenous angiotensin II in the brain was involved in the pain-modulating system. Figure lB shows that a very small dose of saralasin (1 pmol, i.c.v.) did not induced analgesia but did potentiate the as determined analgesic action of morphine (1 nmol, i.c.v.), when injected concomitantly, by the tail-pinch test.
Preliminary
Notes
1133
25
,‘I
1
51016 Conlrol
Figure
I
30
45
60
SO
L
control
minutes after injection
B:
30
80
00
o A .
morphine 2.5 nmol (n= 64), angiotensin II 10 pm01 (n= lo), morphine 2.5 run01 + angiotensin
o A .
morphine 1.0 nmol (n= 24), saralasin 1.0 pm01 (n= 12), + saralasin morphine 1.0 nmol
(B) on morphine-induced
II 10 pmol (n= 16).
1.0 pmol (n= 24).
a: P < 0.01, b: P < 0.05, c: P < 0.10 vs. morphine
Figure
45
minutes after injection
1. Effects of angiotensin II (A) and saralasin analgesia in mice (tail-pinch test). A:
I
16
alone.
2. Dose-dependent attentuation of analgesia induced by morphine (2.5 nmol) by angiotensin II in the tail-pinch test in mice. The columns represent the percentage of the area under the time-course curves for analgesia up to 90 min after the injection (see Fig. l), compared to morphine alone (= 100%). (n) = number of animals.
DISCUSSION Very small doses of angiotensin II (l/250 or less of the dose of morphine, on a molar basis), given intracerebroventricularaly, attenuated the analgesic action of morphine in mice both in the tail-pinch and hot plate tests. This effect must be a form of physiological antagonism, since angiotensin II does not bind to opioid receptors in brain (Keneko, Tamura and Takagi, 1985). The anti-analgesic effect of angiotensin II was longlasting and dose-dependent, but was not complete, even with larger doses of angiotensin II. A very large dose of angiotensin II (1000 pmol) no longer had any effect on the analgesia induced by morphine. This biphasic anti-analgesic effect suggests that the interactions between angiotensin II and morphine involves multiple mechanisms at the different doses of angiotensin II. However, in smaller doses (0.1-10 pmol) angiotensin II is probably selective
1134
Preliminary
Notes
in influencing the mechanism by which morphine produces Its analgesic anti-analgesic effect was apparent without any effect on the Straub's is indicative of an excitatory, non-analgesic effect of morphine.
effect because tail response,
the which
Hyperalgesia was absent after intracerebroventricular administration of angiotensin II (10 pmol), as determined using the hot plate test. Here, the noxious stimulus applied was probably too weak and too short to induce a release of endogenous opioids. In this context, it has been reported that Met-enkephalin is not released from the nucleus reticularis gigantocellularis by acute noxious stimuli, whereas tonic release of Metenkephalin occurs with persistent noxious stimuli (Kuraishi, Sugimoto, Hamada, Kayanoki and Takagi, 1984). The anti-opioid action of endogenous angiotensin II in the brain is strongly supported by the finding that a small dose (1 pmol) of saralasin, given intracerebroventricularly, potentiated the analgesia induced by morphine but did not induce analgesia itself. From these observations, we consider that angiotensin II in the brain has an important role as an anti-opioid substance and is involved in systems modulating opioidfurther mediated pain. In order to clarify the relationship between these neuropeptides, studies on the site and mechanism of action are in progress in this laboratory.
Acknowledgements:
We thank Id. Ohara
(Kyushu University)
for reading
the manuscript.
REFERENCES Elie, R. and Panisset, J-C. (1970). Effect of angiotensin and atropine on the spontaneous release of acetylcholine from cat cerebral cortex. Brain Res., 17:297-305. Ganten, D., Lang, R.E, Lehmann, E. and Ungar, T. (1984). Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem.PharmacoZ.,33:3532-3528. effects produced by intracerebral Haley, T.J. and McCormack, W.G. (1957). Pharmacological injection of drugs in the conscious mouse. Brit.J.PharvnacoZ., 12:12-15. Jhamandas, K., Pinsky, C. and Phillis, J.W. (1970). Effects of morphine on release of cerebral cortical acetylcholine. Nature, 228:176-177.
and its antagonists
Kaneko, S., Tamura, S. and Takagi, Ii. (1985). Purification and identification of endogenous anti-opioid substances from bovine brain. Biochem.Biophys.Res.CollPrmn., 126:587-593. Kuraishi, Y., Sugimoto, Y., Hamada, T., Kayanoki, Y. and Takagi, Ii. (1984). Noxious stimuli and Met-enkephalin release from nucleus reticularis giganticellularis. Brain Res.BuZZ.,
12:123-127. Lee, C-M. and Snyder,
S.H. (1982). Dipeptidyl-aminopeptidase
III of rat brain. J.BioZ.Chem.,
257:12043-12050. Inhibition Stine, f.M., Yang, H-Y.T. and Costa, E. (1980 (Met )-enkephalin and potentiation of (Met b* )-enkephalin Brain Res., 188:295-299.
of in situ metabolism of [3H]analgesia by captopril.
Summy-Long, J.Y., Keil, L.C., Deen, K., Rosella, L. and Severs, W.B. (1981). Endogenous opioid peptide inhibition of the central actions of angiotensin. J.PharmacoZ.Exp.Ther., 217:619-629. Takagi, H., Inukai, T. and Nakama, M. (1966). A modification analgesics. Jpn.J.PharmacoZ.,16:287-294.
of Haffner's
method
for testing
INTRACEREBROVENTRICULAR
ADMINISTRATION
0028-3908/85 $3.00+0.00 Copyright 0 1985PergamonPressLtd
OF ANGIOTENSIN
ANALGESIA S. Kaneko, Department
Faculty
Kyoto University,
IAccepXed
MORPHINE-INDUCED
IN MICE
A. Mori, S. Tamura,
of Pharmacology,
II ATTENUATES
M. Satoh and H. Takagi of Pharmaceutical
Sciences,
Kyoto 606, Japan.
23 Sepmnben.
1985)
Summary - The central effect of angiotensin II on the analgesic action of morphine in mice was investigated using the tail-pinch and hot plate tests. Angiotensin II (0.1-10 pmol), given intracerebroventricularly (i.c.v.), had no effect on the nociceptive sensitivity but did produce a dose-dependent attenuation of the morphine-induced analgesia. A specific angiotensin II antagonist, Saralasin (1 pmol), which in itself had no analgesic effect, significantly potentiated the morphine-induced analgesia. These results suggest that angiotensin II probably plays the physiological role of an anti-opioid substance in pain-modulating systems in the brain.
Endogenous opioid substances, extracted from bovine brain, have been purified and shown to be angiotensin I and II. It was also shown that angiotensin II antagonized the inhibitory effect of Met-enkephalin in the electrically-stimulated guinea-pig ileum preparation, in a physiological manner (Kaneko, Tamura and Takagi, 1985). Angiotensin II has several different pharmacological effects on the central nervous system (Ganten, Lang, Lehmann and Ungar, 1984) and some of these effects are inhibited by opioid peptides (Summy-long, Keil, Deen, Rosella and Severs, 1981). It has been proposed that angiotensins and enkephalins are cleaved by common enzymes in the brain. Angiotensin converting enzyme (EC 3.4.15.1) which converts angiotensin I to angiotensin II, also degrades enkephalin (Stine, Yang and Costa, 1980). Dipeptidyl-aminopeptidase III (EC 3.4.14.4) shows a highly selective affinity for angiotensin EI and enkephalins (Lee and Snyder, 1982). Moreover, earlier studies demonstrated that the output of acetylcholine (ACh 1 from the surface of the cerebral cortex in the cat was decreased by morphine (Jhamandas, Pinsky and Phillis,1970), while it was increased by angiotensin II (Elie and Panisset,lQ?O) However, the actual relationship between angiotensins and opioids in the regulation of pain in the brain is not well understood. brain, mice.
Based on the assumption that angiotensin II may have anti-opioid effects in the the effect of angiotensin II on analgesia induced by morphine was examined in ViVO in
METHODS II (saralasin) were [Ile5]-Angiotensin II and [Sar' ,Val',Ala']-angiotensin purchased from the Protein Research Foundation, Osaka, Japan. These peptides and morphine hydrochloride were dissolved in physiological saline just prior to use. Experiments were carried out on male dd-K mice (15-20 g). Each mouse was given a single intracerebroventricular injection of the drugs in a volume of 10 ~1, using the method of Haley and McCormick (1957). When examining the effect of angiotensin II or saralasin on morphine-induced analgesia, these compounds were mixed together and injected. Analgesia was evaluated by the tail-pinch test (Takagi, Inukai and Nakama, 1966) or the hot plate test. In the tail-pinch test, an aryery clip with a constant pressure of 200 g was used. Mice were used which bit the clip within 2 set after being pinched at the base of the tail, including the anal mucosa, one hour prior to injection. The criterion of analgesia, after the injection of drugs, was an inhibition of the biting response within 6 sec. The results were expressed as the percentage of mice which showed analgesia. In the hot plate test, the mice were placed on a hot stainless steel plate, maintained at 52 f l°C, and the patency of hindpaw-licking or jumping was measured. The baseline latency (To) was first obtained from the mean of two latency values observed 30 and 15 min prior to injection, then the test latency (T) was determined after the injection of drugs, where the cut-off time was 60 sec. The analgesic index (as a percentage [Xl) was calculated as:
?r.P 14
I ,-_I
1131
1132
Preliminary
(T-T0)/(60-To)xlOO.
Notes
The mean f SEM of the To was 21.8tO.4
set (number of animals;
n = 96).
While the analgesic tests were being done, the Straub's tail response, a characteristic excitatory behavioural response of mice, induced by morphine, was also scored. The Straub's tail response was graded by the angle of the tail top to horizontal as: 0°-450 = 0, 45O-90° = 1, more than 90° = 2, at 15, 30, 45, 60 and 90 min after the injection, and the Straub's tail score was determined as the mean of added values of the response per mouse. tail-pinch
Statistical significance was analyzed by Fishers's exact probability test and unpaired t-test (one-tailed) in the hot plate test.
test in the
RESULTS As shown in Figure lA, when angiotensin II (10 pmol) was injected, together with morphine (2.5 nmol)l the analgesic effect of morphine was attenuated for the entire timecourse of the tail-pinch teat, however, the Straub's taile score was not influenced by angiotensin II (group given morphine = 2.53fU.24; group given morphine + angiotensin II = 2.50f0.56). Angiotensin II, when injected alone, did not produced analgesia or abnormal behaviour. Figure 2 summarizes the extent of the suppressant effect of angiotensin II on the analgesia induced by morphine in the tail-pinch test. This effect was dose-dependent in the range 0.1-10 pm01 of angiotensin II. A maximum attenuation was obtained at 10 pmol, but larger doses of angiotensin II were less effective than 10 pmol. A much larger dose of angiotensin II (1000 pmol) had no anti-analgesic effect and no analgesic effect was observed when this dose of angiotensin II was injected alone. In the hot plate test, angiotensin II (10 pmol) alone produced no change in hindpaw-licking or jumping latencies, as compared with the group given saline, but the suppressant effect on morphine-induced analgesia was also observed when angiotensin II was injected together with morphine (Table 1).
Table
1. Effect
of angiotensin
II on morphine-induced
analgesia
in mice
(Hot plate test)
Intracerebroventricular
Minutes
(n)
administration
after injection
15
30
45
60
90
(IO)
-2f8
-4t8
-1f6
-5+8
-6f7
10 pm01
(10)
1+4
-1+6
-1f5
-8k8
-7+9
Morphine
2.5 nmol
(18)
78f8
6'/+9
62+9
43+9
19+4
Morphine + AGT II
2.5 nmol 10 pm01
(18)
48?r9* 45f9*
33t8*
3129
13t8
Saline AGT II
Values
are analgesic
AGT II = angiotensin
index (mean t SEM %).
* P < 0.05 vs. morphine
alone
II.
The effect of saralasin, a specific angiotensin II receptor antagonist, on morphine-induced analgesia was examined in order to determine whether endogenous angiotensin II in the brain was involved in the pain-modulating system. Figure lB shows that a very small dose of saralasin (1 pmol, i.c.v.) did not induced analgesia but did potentiate the as determined analgesic action of morphine (1 nmol, i.c.v.), when injected concomitantly, by the tail-pinch test.
Preliminary
Notes
1133
25
,‘I
1
51016 Conlrol
Figure
I
30
45
60
SO
L
control
minutes after injection
B:
30
80
00
o A .
morphine 2.5 nmol (n= 64), angiotensin II 10 pm01 (n= lo), morphine 2.5 run01 + angiotensin
o A .
morphine 1.0 nmol (n= 24), saralasin 1.0 pm01 (n= 12), + saralasin morphine 1.0 nmol
(B) on morphine-induced
II 10 pmol (n= 16).
1.0 pmol (n= 24).
a: P < 0.01, b: P < 0.05, c: P < 0.10 vs. morphine
Figure
45
minutes after injection
1. Effects of angiotensin II (A) and saralasin analgesia in mice (tail-pinch test). A:
I
16
alone.
2. Dose-dependent attentuation of analgesia induced by morphine (2.5 nmol) by angiotensin II in the tail-pinch test in mice. The columns represent the percentage of the area under the time-course curves for analgesia up to 90 min after the injection (see Fig. l), compared to morphine alone (= 100%). (n) = number of animals.
DISCUSSION Very small doses of angiotensin II (l/250 or less of the dose of morphine, on a molar basis), given intracerebroventricularaly, attenuated the analgesic action of morphine in mice both in the tail-pinch and hot plate tests. This effect must be a form of physiological antagonism, since angiotensin II does not bind to opioid receptors in brain (Keneko, Tamura and Takagi, 1985). The anti-analgesic effect of angiotensin II was longlasting and dose-dependent, but was not complete, even with larger doses of angiotensin II. A very large dose of angiotensin II (1000 pmol) no longer had any effect on the analgesia induced by morphine. This biphasic anti-analgesic effect suggests that the interactions between angiotensin II and morphine involves multiple mechanisms at the different doses of angiotensin II. However, in smaller doses (0.1-10 pmol) angiotensin II is probably selective
1134
Preliminary
Notes
in influencing the mechanism by which morphine produces Its analgesic anti-analgesic effect was apparent without any effect on the Straub's is indicative of an excitatory, non-analgesic effect of morphine.
effect because tail response,
the which
Hyperalgesia was absent after intracerebroventricular administration of angiotensin II (10 pmol), as determined using the hot plate test. Here, the noxious stimulus applied was probably too weak and too short to induce a release of endogenous opioids. In this context, it has been reported that Met-enkephalin is not released from the nucleus reticularis gigantocellularis by acute noxious stimuli, whereas tonic release of Metenkephalin occurs with persistent noxious stimuli (Kuraishi, Sugimoto, Hamada, Kayanoki and Takagi, 1984). The anti-opioid action of endogenous angiotensin II in the brain is strongly supported by the finding that a small dose (1 pmol) of saralasin, given intracerebroventricularly, potentiated the analgesia induced by morphine but did not induce analgesia itself. From these observations, we consider that angiotensin II in the brain has an important role as an anti-opioid substance and is involved in systems modulating opioidfurther mediated pain. In order to clarify the relationship between these neuropeptides, studies on the site and mechanism of action are in progress in this laboratory.
Acknowledgements:
We thank Id. Ohara
(Kyushu University)
for reading
the manuscript.
REFERENCES Elie, R. and Panisset, J-C. (1970). Effect of angiotensin and atropine on the spontaneous release of acetylcholine from cat cerebral cortex. Brain Res., 17:297-305. Ganten, D., Lang, R.E, Lehmann, E. and Ungar, T. (1984). Brain angiotensin: on the way to becoming a well-studied neuropeptide system. Biochem.PharmacoZ.,33:3532-3528. effects produced by intracerebral Haley, T.J. and McCormack, W.G. (1957). Pharmacological injection of drugs in the conscious mouse. Brit.J.PharvnacoZ., 12:12-15. Jhamandas, K., Pinsky, C. and Phillis, J.W. (1970). Effects of morphine on release of cerebral cortical acetylcholine. Nature, 228:176-177.
and its antagonists
Kaneko, S., Tamura, S. and Takagi, Ii. (1985). Purification and identification of endogenous anti-opioid substances from bovine brain. Biochem.Biophys.Res.CollPrmn., 126:587-593. Kuraishi, Y., Sugimoto, Y., Hamada, T., Kayanoki, Y. and Takagi, Ii. (1984). Noxious stimuli and Met-enkephalin release from nucleus reticularis giganticellularis. Brain Res.BuZZ.,
12:123-127. Lee, C-M. and Snyder,
S.H. (1982). Dipeptidyl-aminopeptidase
III of rat brain. J.BioZ.Chem.,
257:12043-12050. Inhibition Stine, f.M., Yang, H-Y.T. and Costa, E. (1980 (Met )-enkephalin and potentiation of (Met b* )-enkephalin Brain Res., 188:295-299.
of in situ metabolism of [3H]analgesia by captopril.
Summy-Long, J.Y., Keil, L.C., Deen, K., Rosella, L. and Severs, W.B. (1981). Endogenous opioid peptide inhibition of the central actions of angiotensin. J.PharmacoZ.Exp.Ther., 217:619-629. Takagi, H., Inukai, T. and Nakama, M. (1966). A modification analgesics. Jpn.J.PharmacoZ.,16:287-294.
of Haffner's
method
for testing