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Possible involvement of μ2-opioid receptor-mediated mechanisms in morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex in the mouse
Life Sciences, Vol. 59, No. 22,pp. PL 349-353,19% CopyTight0 19% Ehvier science Inc. Printed in the WA. All rights reserved 0024-3205/% $15.00 + .oa
ELSEVIER
PI1 SOO24-3205(96)00540-1
Ph%RMXCOLOGY LElTERS Accelemted Communication
POSSIBLE INVOLVEMENT OF k-OPIOID RECEPTOR-MEDIATED MECHANISMS IN MORPHINE-INDUCED ENHANCEMENT OF THE PENTOBARBITAL-INDUCED LOSS OF THE RIGHTING REFLEX IN THE MOUSE Junzo Kamei, Masahiro Ohsawa and Hiroshi Nagase * Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo 142, Japan. *Basic Research Laboratories, Toray Industries, Kamakura 248, Japan. (Submitted July 31, 19%; accepted August 15, 1996; received in hal form September 18, 19%)
Abstract: The effects of pretreatment with selective p-, & and k-opioid receptor antagonists on the enhancement of the pentobarbital-induced loss of the righting reflex by morphine were examined in mice. Mice injected with pentobarbital(50 mgkg, i.p.) showed an increased duration for the loss of the righting reflex after administration of morphine. This enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine (10 mg/kg, s.c.) was significantly antagonized by pretreatment with flfunaltrexamine, a selective p-opioid receptor antagonist, but not by pretreatment with naloxonazine, a selective pl-opioid receptor antagonist. Furthermore, neither naltrindole, a selective &opioid receptor antagonist, nor nor-binaltorphimine, a selective rc-opioid receptor antagonist, had a significant effect on the enhancement of the pentobarbitalinduced loss of the righting reflex by morphine. These results suggest that k-, rather than pl-, 6- and K-, opioid receptor-mediated mechanisms are involved in the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. Key Wwdc
pentobarbital, morphine, sleeping time, PI-opioid receptor, rz-opioid receptor, naloxonazine
Introduction Potentiation of the effects of barbiturates by morphine is not a new observation (l-5), but the mechanism of this potentiation is still unclear. Previous studies have concluded that the enhancement of barbiturate-induced sleeping time produced by morphine is due to inhibition of hepatic drug-metabolizing activity (1,3,4); moreover, it has been reported that this interaction involves opiate receptors (6). However, the involvement of opioid receptor subtypes in the morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex has not been studied. Therefore, in the present study, we examined the effects of antagonists for each opioid receptor type, i.e., p-, h-, & and tc-opioid receptors, on the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. Correspondence: J. Kamei, Ph.D., Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan. FAX: 81-3-5498-5029
PL350
Pentobarbital-induced Sleeping and Morphine
Vol. 59, No. 22,1996
Materials and Methods Animals
Male ICR mice (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan), weighing about 20 g at the beginning of the experiments, were used. They had free access to food and water in an animal room with a 12-hour light-dark cycle. Measurement of the Duration of the loss of the RiRhtinR Reflex The duration of the loss of the righting response was measured
according to the procedures described by Marley et al. (7). Mice were given an i.p. injection of pentobarbital(50 mg/kg). When the animals become ataxic, they were placed in a V-shape plastic trough and the time was recorded. Animals were judged to have regained the righting response when they could right themselves three times within 30 sec. The amount of time between the loss and regaining of the response was rounded to the nearest minute.
Data analysis Data were expressed as the mean with SE. Statistical significance of differences was assessed by Student’s t-test (comparison of two groups) or a one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett’s test (comparison of multiple group). A level of probability of 0.05 or less was accepted as significant.
Sodium pentobarbital was purchased from Tokyo Kasei Kogyo, Co., Tokyo, Japan. Morphine hydrochloride was purchased from Sankyo, Co., Tokyo, Japan. l3Funaltrexamine (FNA), naloxonazine (NXZ), naltrindole (NTI) and nor-binaltorphimine (BNI) were synthesized by Dr. Nagase (Toray Industries, Kamakura, Japan). All drugs were dissolved in 0.9% saline solution. Morphine (l-10 mg/kg, s.c.) injection was immediately followed by an injection of sodium pentobarbital(50 mg/kg, i.p.). FNA (20 mg/kg, s.c.) and NXZ (35 mgkg, s.c.) were administered 24 hr before the injection of morphine. NTI (1 mgkg, s.c.) was administered 15 min before the injection of morphine. Nor-binaltorphimine (20 mg/kg, s.c.) was administered 3 hr before the injection of morphine. The dose and schedule for each opioid antagonist in this study were determined as described previously (8).
Results As shown in Fig. 1, subcutaneous injection of morphine at doses of 1, 3, 10 and 30 mg/kg produced dose-dependent increases in the duration of the pentobarbital-induced loss of the righting reflex. Morphine at doses of 10 and 30 mg/kg significantly increased the mean duration of the pentobarbital-induced loss of the righting reflex. However, the effect of morphine was not dose-dependent between 10 and 30 mg/kg. As shown in Fig. 2, when mice were pretreated with FNA, the morphine-induced enhancement of the duration of the pentobarbital-induced loss of the righting reflex was significantly reduced. However, the ability of morphine to enhance the duration of the pentobarbital-induced loss of the righting reflex was unchanged after pretreatment with either ,NT’Ior BNI (Fig. 2). On the other hand, the morphine-induced enhancement of the duration of the pentobarbital-induced loss of righting reflex was significantly enhanced by pretreatment with NXZ.
Vol. 59, No. 22, 1996
Pentobarbital-induced Sleeping and Morphine
PL351
z
flO0 G=i
!2
*
*
10
30
2
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r:
50
;
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2 6
0 Saline
3
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Morphine Pentobarbital
(mg/kg, s.c.)
(50 mg/kg, i.p.)
Fig. 1. The effect of morphine on the pentobarbital (50 mgkg, i.p.)-induced loss of the righting reflex in mice. Each column represents the mean with S.E. of 10 mice. *P
Discussion In the present study, we demonstrated that morphine produced dose-related enhancement of the duration of the pentobarbital-induced loss of the righting reflex in mice. We previously reported that morphine at doses of 10, 20 and 40 mgkg, s.c., had a dose-dependent locomotor-enhancing effect in mice (9). In the present study, indeed, morphine at doses of 10 and 30 mg/kg produced neither catalepsy nor motor impairment. Thus, It is possible that morphine-induced enhancement of pentobarbital-induced loss of righting reflex is not due to the morphine-induced alternations in motor competency, such as catalepsy and motor impairment. Furthermore, this morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex was antagonized by S.C.pretreatment with FNA, a selective p-opioid receptor antagonist. This finding is consistent with a previous report that pretreatment with naltrexone, a p-opioid receptor antagonist, completely blocked the acute morphine-induced enhancement of pentobarbital-induced sleeping time (6). Furthermore, Fatherazi et al. (10) reported that central injection of morphine into the septum increased the duration of pentobarbital-induced sleep, and this effect was antagonized by injections of naloxone. These results and the present data strongly support the possibility that the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex is mediated by opioid receptors, rather than by nonspecific effects of morphine. In the present study, we also observed that neither NTI, a selective &opioid receptor antagonist, nor BNI, a selective k-opioid receptor antagonist, had a significant effect on the
Vol. 59, No. 22, 19%
Pentobarbital-inducedSleeping and Morphine
PL-352
morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. This result indicates that the enhancement of the duration of the pentobarbital-induced loss of the righting reflex produced by morphine results from the activation of p-, but not of 6or K-, opioid receptors. Moreover, naloxonazine, a selective pr-opioid receptor antagonist, did not antagonize the morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex. Therefore, it is possible that naloxonazine-insensitive p-opioid receptors, namely k-opioid receptors, are responsible for the enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine. An unexpected finding in this study is that the effect of morphine on the pentobarbitalinduced loss of the righting reflex was significantly enhanced by NXZ. The mechanism leading to the enhancement of the effect of morphine on the pentobarbital-induced loss of the righting reflex by NXZ is unclear. One possible speculation for this phenomenon is that the blockade of pl-opioid receptors by NXZ might modify the h-opioid receptor-effector coupling mechanisms or modify the binding of morphine to k-opioid receptors. However, further studies are necessary before this possibility can be established. In conclusion, the enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine may be mediated mainly though the activation of h-opioid receptors.
Y3 &oo 5 G z 2 ‘;: _c +? 50 ru 0
z 0 ?I g ‘;:
2
8
0 Saline Saline FNA
NXZ
NTI
BNI
Morphine (10 mg/kg, s.c.) Pentobarbital (50 mgkg, i.p.) Fig. 2.
Blockade of the morphine-induced enhancement of the pentobarbitalinduced loss of the righting reflex by opioid receptor antagonists. @Funaltrexamine (FNA, 20 mg_/kg)and naloxonazine (NXZ, 35 mg/kg) were injected S.C. 24 h before morphine administration. Naltrindole (NIT, 1 mg/kg) was injected S.C. 15 min before morphine administration. Norbinaltorphimine @II, 20 mg/kg) was injected S.C. 3 h before morphine administration. Each column represents the mean with S.E. of 10 mice. *P
Vol. 59, No. 22, 19%
Pentobarbital-induced Sleeping and Morphine
PL-353
Acknowledgements We would like to thank Ms. Y. Tamotsu, Ms. M. Itoh, Ms. M. Kobayashi Sawada for their technical assistance.
and Ms. C.
References 1. W.F. BOUSQUET, B.D. RUPE and T.S. MIYA, Biochem. Pharmacol., 13 123-125 (1964). 2. B.D. CHERKSEY and N. ALTSZULER, Pharmacology, 12 362-371 (1974). 3. I.K. HO, I. YAMAMOTO, ICE. BECKER, H.H. LOH and E.L. WAY, Life Sci., 19 357366 (1976). 4. I.K. HO, I. YAMAMOTO, H.H. LOH and E.L.WAY,Biochem. Pharmacol., -25 357-358 (1976). 5. R.A. Howd and G.T. Pryor, Pharmacol. Biochem. Behav., 12 577-586 (1980). 6. R.N. PECHINICK and G.W. TERMAN, Neuropharmacoiogy, 26 1589-1593 (1986). 7. R.J. MARLEY, L.L. MINER, J.M. WEHNER, and A.C. COLLINS, J. Pharmacol. Exp. Ther., 238 1028-1033 (1986). 8. J. KAMEI, T. SUZUKI, M. MISAWA, H. NAGASE and Y. KASUYA Eur. J. Pharmacol., 275 109-113 (1995). 9 J.-I, M. OHSAWA, A. SAITOH, Y. IWAMOTO, T. SUZUKI, M. MISAWA, H. NAGASE, and Y. KASUYA, J. Pharmacol. Exp. Ther.,m 700-706 (1995) 10. S. FATHERAZI, H. LAI, S. KAZI and A. HORITA, Pharmacol. Biochem. Behav., 23 505-507 (1985).
ELSEVIER
PI1 SOO24-3205(96)00540-1
Ph%RMXCOLOGY LElTERS Accelemted Communication
POSSIBLE INVOLVEMENT OF k-OPIOID RECEPTOR-MEDIATED MECHANISMS IN MORPHINE-INDUCED ENHANCEMENT OF THE PENTOBARBITAL-INDUCED LOSS OF THE RIGHTING REFLEX IN THE MOUSE Junzo Kamei, Masahiro Ohsawa and Hiroshi Nagase * Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo 142, Japan. *Basic Research Laboratories, Toray Industries, Kamakura 248, Japan. (Submitted July 31, 19%; accepted August 15, 1996; received in hal form September 18, 19%)
Abstract: The effects of pretreatment with selective p-, & and k-opioid receptor antagonists on the enhancement of the pentobarbital-induced loss of the righting reflex by morphine were examined in mice. Mice injected with pentobarbital(50 mgkg, i.p.) showed an increased duration for the loss of the righting reflex after administration of morphine. This enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine (10 mg/kg, s.c.) was significantly antagonized by pretreatment with flfunaltrexamine, a selective p-opioid receptor antagonist, but not by pretreatment with naloxonazine, a selective pl-opioid receptor antagonist. Furthermore, neither naltrindole, a selective &opioid receptor antagonist, nor nor-binaltorphimine, a selective rc-opioid receptor antagonist, had a significant effect on the enhancement of the pentobarbitalinduced loss of the righting reflex by morphine. These results suggest that k-, rather than pl-, 6- and K-, opioid receptor-mediated mechanisms are involved in the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. Key Wwdc
pentobarbital, morphine, sleeping time, PI-opioid receptor, rz-opioid receptor, naloxonazine
Introduction Potentiation of the effects of barbiturates by morphine is not a new observation (l-5), but the mechanism of this potentiation is still unclear. Previous studies have concluded that the enhancement of barbiturate-induced sleeping time produced by morphine is due to inhibition of hepatic drug-metabolizing activity (1,3,4); moreover, it has been reported that this interaction involves opiate receptors (6). However, the involvement of opioid receptor subtypes in the morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex has not been studied. Therefore, in the present study, we examined the effects of antagonists for each opioid receptor type, i.e., p-, h-, & and tc-opioid receptors, on the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. Correspondence: J. Kamei, Ph.D., Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan. FAX: 81-3-5498-5029
PL350
Pentobarbital-induced Sleeping and Morphine
Vol. 59, No. 22,1996
Materials and Methods Animals
Male ICR mice (Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan), weighing about 20 g at the beginning of the experiments, were used. They had free access to food and water in an animal room with a 12-hour light-dark cycle. Measurement of the Duration of the loss of the RiRhtinR Reflex The duration of the loss of the righting response was measured
according to the procedures described by Marley et al. (7). Mice were given an i.p. injection of pentobarbital(50 mg/kg). When the animals become ataxic, they were placed in a V-shape plastic trough and the time was recorded. Animals were judged to have regained the righting response when they could right themselves three times within 30 sec. The amount of time between the loss and regaining of the response was rounded to the nearest minute.
Data analysis Data were expressed as the mean with SE. Statistical significance of differences was assessed by Student’s t-test (comparison of two groups) or a one-way analysis of variance (ANOVA) with repeated measures followed by Dunnett’s test (comparison of multiple group). A level of probability of 0.05 or less was accepted as significant.
Sodium pentobarbital was purchased from Tokyo Kasei Kogyo, Co., Tokyo, Japan. Morphine hydrochloride was purchased from Sankyo, Co., Tokyo, Japan. l3Funaltrexamine (FNA), naloxonazine (NXZ), naltrindole (NTI) and nor-binaltorphimine (BNI) were synthesized by Dr. Nagase (Toray Industries, Kamakura, Japan). All drugs were dissolved in 0.9% saline solution. Morphine (l-10 mg/kg, s.c.) injection was immediately followed by an injection of sodium pentobarbital(50 mg/kg, i.p.). FNA (20 mg/kg, s.c.) and NXZ (35 mgkg, s.c.) were administered 24 hr before the injection of morphine. NTI (1 mgkg, s.c.) was administered 15 min before the injection of morphine. Nor-binaltorphimine (20 mg/kg, s.c.) was administered 3 hr before the injection of morphine. The dose and schedule for each opioid antagonist in this study were determined as described previously (8).
Results As shown in Fig. 1, subcutaneous injection of morphine at doses of 1, 3, 10 and 30 mg/kg produced dose-dependent increases in the duration of the pentobarbital-induced loss of the righting reflex. Morphine at doses of 10 and 30 mg/kg significantly increased the mean duration of the pentobarbital-induced loss of the righting reflex. However, the effect of morphine was not dose-dependent between 10 and 30 mg/kg. As shown in Fig. 2, when mice were pretreated with FNA, the morphine-induced enhancement of the duration of the pentobarbital-induced loss of the righting reflex was significantly reduced. However, the ability of morphine to enhance the duration of the pentobarbital-induced loss of the righting reflex was unchanged after pretreatment with either ,NT’Ior BNI (Fig. 2). On the other hand, the morphine-induced enhancement of the duration of the pentobarbital-induced loss of righting reflex was significantly enhanced by pretreatment with NXZ.
Vol. 59, No. 22, 1996
Pentobarbital-induced Sleeping and Morphine
PL351
z
flO0 G=i
!2
*
*
10
30
2
‘S
r:
50
;
z
L! %
s
‘5
2 6
0 Saline
3
1
Morphine Pentobarbital
(mg/kg, s.c.)
(50 mg/kg, i.p.)
Fig. 1. The effect of morphine on the pentobarbital (50 mgkg, i.p.)-induced loss of the righting reflex in mice. Each column represents the mean with S.E. of 10 mice. *P
Discussion In the present study, we demonstrated that morphine produced dose-related enhancement of the duration of the pentobarbital-induced loss of the righting reflex in mice. We previously reported that morphine at doses of 10, 20 and 40 mgkg, s.c., had a dose-dependent locomotor-enhancing effect in mice (9). In the present study, indeed, morphine at doses of 10 and 30 mg/kg produced neither catalepsy nor motor impairment. Thus, It is possible that morphine-induced enhancement of pentobarbital-induced loss of righting reflex is not due to the morphine-induced alternations in motor competency, such as catalepsy and motor impairment. Furthermore, this morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex was antagonized by S.C.pretreatment with FNA, a selective p-opioid receptor antagonist. This finding is consistent with a previous report that pretreatment with naltrexone, a p-opioid receptor antagonist, completely blocked the acute morphine-induced enhancement of pentobarbital-induced sleeping time (6). Furthermore, Fatherazi et al. (10) reported that central injection of morphine into the septum increased the duration of pentobarbital-induced sleep, and this effect was antagonized by injections of naloxone. These results and the present data strongly support the possibility that the morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex is mediated by opioid receptors, rather than by nonspecific effects of morphine. In the present study, we also observed that neither NTI, a selective &opioid receptor antagonist, nor BNI, a selective k-opioid receptor antagonist, had a significant effect on the
Vol. 59, No. 22, 19%
Pentobarbital-inducedSleeping and Morphine
PL-352
morphine-induced enhancement of the pentobarbital-induced loss of the righting reflex. This result indicates that the enhancement of the duration of the pentobarbital-induced loss of the righting reflex produced by morphine results from the activation of p-, but not of 6or K-, opioid receptors. Moreover, naloxonazine, a selective pr-opioid receptor antagonist, did not antagonize the morphine-mediated enhancement of the pentobarbital-induced loss of the righting reflex. Therefore, it is possible that naloxonazine-insensitive p-opioid receptors, namely k-opioid receptors, are responsible for the enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine. An unexpected finding in this study is that the effect of morphine on the pentobarbitalinduced loss of the righting reflex was significantly enhanced by NXZ. The mechanism leading to the enhancement of the effect of morphine on the pentobarbital-induced loss of the righting reflex by NXZ is unclear. One possible speculation for this phenomenon is that the blockade of pl-opioid receptors by NXZ might modify the h-opioid receptor-effector coupling mechanisms or modify the binding of morphine to k-opioid receptors. However, further studies are necessary before this possibility can be established. In conclusion, the enhancement of the pentobarbital-induced loss of the righting reflex produced by morphine may be mediated mainly though the activation of h-opioid receptors.
Y3 &oo 5 G z 2 ‘;: _c +? 50 ru 0
z 0 ?I g ‘;:
2
8
0 Saline Saline FNA
NXZ
NTI
BNI
Morphine (10 mg/kg, s.c.) Pentobarbital (50 mgkg, i.p.) Fig. 2.
Blockade of the morphine-induced enhancement of the pentobarbitalinduced loss of the righting reflex by opioid receptor antagonists. @Funaltrexamine (FNA, 20 mg_/kg)and naloxonazine (NXZ, 35 mg/kg) were injected S.C. 24 h before morphine administration. Naltrindole (NIT, 1 mg/kg) was injected S.C. 15 min before morphine administration. Norbinaltorphimine @II, 20 mg/kg) was injected S.C. 3 h before morphine administration. Each column represents the mean with S.E. of 10 mice. *P
Vol. 59, No. 22, 19%
Pentobarbital-induced Sleeping and Morphine
PL-353
Acknowledgements We would like to thank Ms. Y. Tamotsu, Ms. M. Itoh, Ms. M. Kobayashi Sawada for their technical assistance.
and Ms. C.
References 1. W.F. BOUSQUET, B.D. RUPE and T.S. MIYA, Biochem. Pharmacol., 13 123-125 (1964). 2. B.D. CHERKSEY and N. ALTSZULER, Pharmacology, 12 362-371 (1974). 3. I.K. HO, I. YAMAMOTO, ICE. BECKER, H.H. LOH and E.L. WAY, Life Sci., 19 357366 (1976). 4. I.K. HO, I. YAMAMOTO, H.H. LOH and E.L.WAY,Biochem. Pharmacol., -25 357-358 (1976). 5. R.A. Howd and G.T. Pryor, Pharmacol. Biochem. Behav., 12 577-586 (1980). 6. R.N. PECHINICK and G.W. TERMAN, Neuropharmacoiogy, 26 1589-1593 (1986). 7. R.J. MARLEY, L.L. MINER, J.M. WEHNER, and A.C. COLLINS, J. Pharmacol. Exp. Ther., 238 1028-1033 (1986). 8. J. KAMEI, T. SUZUKI, M. MISAWA, H. NAGASE and Y. KASUYA Eur. J. Pharmacol., 275 109-113 (1995). 9 J.-I, M. OHSAWA, A. SAITOH, Y. IWAMOTO, T. SUZUKI, M. MISAWA, H. NAGASE, and Y. KASUYA, J. Pharmacol. Exp. Ther.,m 700-706 (1995) 10. S. FATHERAZI, H. LAI, S. KAZI and A. HORITA, Pharmacol. Biochem. Behav., 23 505-507 (1985).