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Inhibition by ginseng total saponin of the development of morphine reverse tolerance and dopamine receptor supersensitivity in mice
Pergamon
0306-3623(94)00267-3
Gen. Pharmac.Vol. 26, No. 5, pp. 1071-1076, 1995 Copyright© 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/95 $9.50+ 0.00
Inhibition by Ginseng Total Saponin of the Development of Morphine Reverse Tolerance and Dopamine Receptor Supersensitivity in Mice HACK-Seang KIM,* JIN-Goo KANG and KI-Wan OH Department of Pharmacology, College of Pharmacy, Chungbuk National University, Cheong/u 360-763, Korea [Fax: (431) 68-27321 (Received 13 September 1994)
Abstraet--l. Ginseng total saponin (GTS), 200mg/kg i.p. 3hr prior to morphine, inhibited the development of reverse tolerance to the ambulatory-accelerating effect of morphine. 2. GTS, 200 mg/kg, also prevented the development of dopamine receptor supersensitivity induced by the chronic administration of morphine, 10 mg/kg a day for 7 days. 3. These results suggest that GTS may be useful for the prevention and therapy of the adverse action of morphine.
Key Words: Ginseng total saponin (GTS), reverse tolerance, morphine, supersensitivity
INTRODUCTION The pharmacological effects of ginseng on various organs have been reviewed (Katu et al., 1975; Saito et al., 1973). Petkov (1959) demonstrated that ginseng extract had a variety of effects on the activity of the central nervous system (CNS). Ginseng not only stimulates in low dose but also inhibits the CNS (Takagi et al., 1972). It also acts as a modulator of neurotransmission in the brain (Kim et al., 1985; Tsang et al., 1985). Dopaminergic and noradrenergic neurons in the CNS play important roles in the behavioral effects of drugs (Dwoskin et al., 1988; Porrino et al., 1988; Puri and Lal, 1973; Taylor et al., 1979; Wood and Altar, 1988). The repeated intermittent administration of CNS stimulants to animals produces progressive changes in behavior, a phenomenon known as behavioral sensitization (reverse tolerance) (Hirabayashi and Alam, 1981; Kilbey and Ellinwood, 1977; Kuribara and Tadokoro, 1989; Mansour et aL, 1987; Shuster et al., 1975). It is thought that this sensitization represents an animal model of drug*To whom all correspondence should be addressed.
induced psychosis (Allen and Young, 1978; Robinson and Becker, 1986). It is also reported that rats sensitized to morphine show an enhanced response to apomorphine, a direct dopamine receptor agonist, suggesting the development of dopamine receptor supersensitivity (Bhargava, 1980; Ritzmann et al., 1979). Behavioral sensitization after the repeated administration of morphine has been demonstrated to be attributable to the dopaminergic hyperfunction in the CNS (Kilbey and Ellinwood, 1977; Puri and Lal, 1973; Robinson and Becker, 1986; Taylor et al., 1979). On the other hand, Bhargava and Ramarao (1991) reported that ginseng extract had an inhibitory activity on the development of tolerance to the pharmacological actions of morphine. In addition, Tokuyama et al. (1992) have shown that the standardized ginseng extract prevents the development of reverse tolerance to the ambulation-accelerating effect of methamphetamine. Therefore, the present experiments were undertaken to determine the effects of GTS, as an active component fraction of ginseng extract, on the development of reverse tolerance to the ambulationaccelerating effect of morphine in mice. The effects
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of GTS on the dopamine receptor supersensitivity induced by morphine were also examined. MATERIALS AND M E T H O D S
Animals and drugs ICR male mice weighing 20-30 g in a group of 10-12, were used in all experiments. They were housed in the acrylic fiber cage in a controlled room (22 ___I°C), and were kept on unlimited supply of the solid diet and tap water. The drugs used were morphine hydrochloride (Dae-Won Pharm. Co., Korea), apomorphine hydrochloride (Sigma, U.S.A.) and GTS [characterized saponins mixture containing at least 10 glycosides Rb 2 (18.26%), Rb2 (9.07), Rc (9.65%), Rd (8.24%), Re (9.28%), Rf (3.48%), Rgl (6.42%), Rg 2 (3.62%), Rg 3 (4.70%), Ro (3.82%), Ra (2.91%) and minor ginsenosides and components (20.55%) from panax ginseng, extracted and purified by Namba et al.'s method (Namba et al., 1974) and supplied by Korea Ginseng and Tobacco Research Institute]. Except for apomorphine, the drugs were dissolved in physiological saline. Apomorphine was dissolved in saline containing 0.1% ascorbic acid, just prior to the experiment. Morphine was administered to mice subcutaneously (s.c.), and GTS and apomorphine were administered to mice intraperitoneally (i.p.).
Measurement of the development of reverse tolerance to the ambulation-accelerating effect As the development of "reverse tolerance" is subject to such variations as drug dosage, route of administration and duration of treatment, for each endpoint investigated, parameters were selected empirically to produce a significant degree of reverse tolerance within a period of 7 days or less. For example, the rate of development of reverse tolerance is generally dependent on the amount of dosage, therefore, the daily drug dosage was designed to produce a significant change in endpoint within the limited duration of the study. Furthermore, in mouse, reverse tolerance to morphine develops more expeditiously to s.c. than i.p. administration, hence the former route of administration was used to produce reverse tolerance in these experiments. The ambulatory activity of mice was measured by the tilting-type ambulometer (AMB-10, O'Hara & Co., Japan). Each mouse was placed in activity cages of 20 cm in diameter and 18 cm in height. Drug administration was carried out after an adaptation period of 10rain. Daily ambulatory activity was measured for 3 hr after morphine administration. Morphine 10 mg/kg was administered once per day for 7 days. The development of reverse
tolerance was evidenced by the enhanced ambulationaccelerating activity with the repeated administration of morphine. When the combined effect of morphine and GTS was investigated at various time intervals, the consistent and reliable inhibitory effects of GTS were observed in mice pretreated with GTS 3 hr prior to morphine administration. Therefore, mice were pretreated with GTS 100 or 200 mg/kg 3 hr prior to morphine injection.
Measurement of the development of dopamine receptor supersensitivity The development of dopamine receptor supersensitivty was determined by measuring the enhanced hypothermic response to and the enhanced ambulatory activity of apomorphine (a dopaminergic receptor agonist) in mice treated with morphine. Additional groups of mice with the same chronic morphine and GTS treatment, as in the measurement of the development of reverse tolerance in the previous test, were used to determine the effects of these treatments on the hypothermic response to apomorphine. The hypothermic response to an i.p. injection of apomorphine 1 mg/kg (Ritzmann, 1979), a dosage sufficient to produce hypothermia, was determined 24 hr after the final injection of morphine. Body temperature was determined by using a rectal probe (inserted 2.5 cm into the rectum) and a telethermometer. The measurements were made just prior to and 30 min after apomorphine injection. Data were expressed as the difference between the measurements before and after the injection of apomorphine. Groups of mice with the same chronic morphine and GTS treatment as in the previous test, were used to determine the effects of these treatments on the apomorphine induced ambulation-accelerating activity. The ambulationaccelerating activity of apomorphine was measured 24 hr after the final injection of morphine. Mice were first allowed to preambulate for 10min followed by a 20min test period. After an interval of 30min, mice were given apomorphine 4mg/kg, a dosage which produced significant increase of ambulatory activity (Bhargava, 1980). Immediately after administration, the 10min preambulation period and 20 min test period were repeated. Data were expressed as the difference between the test activity counts before and after the injection of apomorphine.
Statistics The data expressed were as mean _ SE. The significant differences were first analyzed by various (ANOVA). In case of significant variation, the individual values were compared by Student's t-test.
Morphine and ginseng 3000
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1073
tration of the drugs with 2600 counts on day 6, compared with 1370 counts on day 1. GTS given 3 hr before morphine injection, dose-dependently suppressed the development of reverse tolerance to morphine. The activity count on day 6, was approx. 1400, about 1200 less than that of the morphine pretreated group in mice pretreated with GTS 200 mg/kg, (Fig. 1). These results suggest that GTS inhibits the development of reverse tolerance to the ambulation-accelerating effects of morphine.
Inhibitory effects of GTS on the development of dopamine receptor supersensitivity 1
2
3
4
5
8
?
Fig. I. Effect of GTS on the development of reverse tolerance to the ambulation-accelerating effect of morphine. Morphine I0 mg/kg (s.c.) was administered to mice once a day for 7 days. GTS I00 or 200 mg/kg (i.p.) was administered to mice 3 hr prior to the injection of morphine. The ambulatory activity was measured for 1 hr after morphine administration. 4fP < 0.05; # # P < 0.01, compared with the count on day I. *P <0.05; **P <0.01, compared with that of the morphine group. © ©: Saline; • O: morphine 10 mg/kg; A A: GTS 100 mg/kg; V V: GTS 200 mg/kg; O O: morphine 10mg/kg+GTS 100mg/kg; and [] I:]: morphine l0 mg/kg + GTS 200 mg/kg.
RESULTS
Inhibitory effects of GTS on the development of morphine induced reverse tolerance to the ambulationaccelerating effect Figure 1 shows the mean overall ambulatory activity after the repeated administration of morphine (10 mg/kg). The ambulation-accelerating activities of morphine were enhanced by repeated treatment, i.e. reverse tolerance was developed to the ambulation-accelerating effects of morphine. Single or chronic treatment with GTS or saline alone did not influence the spontaneous motor activity of mice. Morphine induced ambulation-accelerating activity was markedly enhanced by the repeated adminis-
The hypothermic response to apomorphine I mg/kg was enhanced in mice treated with morphine, in comparison to the saline treatment group. Morphine treated mice receiving chronic injection of GTS 100 or 200mg/kg 3 hr prior to the injection of morphine did not exhibit an enhanced hypothermic response to apomorphine (Table 1). Single or chronic treatment with GTS or saline alone did not influence the hypothermic response of mice. The enhanced hypothermic response of apomorphine in chronic morphine treated mice was inhibited by GTS 200 mg/kg pretreatment 3 hr prior to the injection of morphine (Table 1). Apomorphine at a dose of 4 mg/kg produced a significant increase of ambulatory activity in chronic treated mice, compared with the increase observed in saline treated mice. The enhanced ambulatory activity of apomorphine in chronic morphine treated mice was inhibited by GTS 200 mg/kg pretreatment 3 hr prior to the injection of morphine (Fig. 2). These results make it evident that chronic administration of morphine develops dopamine receptor supersensitivity, and that GTS blocks the development of dopamine receptor supersensitivity. DISCUSSION It has been reported that the standardized ginseng extract inhibited the development of reverse tolerance
Table I. Effect o f G T S on the enhanced hypothermic response to apomorphine in mice treated with m o r p h i n e
Body temperature(°C) Group Saline G T S 200 Saline + m o r p h i n e G T S 100 + morphine G T S 200 + m o r p h i n e
At 0 rain 38.20 38.23 38.14 38.24 38.06
4- 0.24 +_ 0.22 4- 0.10 -4- 0.10 4- 0.13
At 30 min 37.21 37.28 36.65 36.99 36.93
_ 0.10 4- 0.17 _ 0.11 __ 0.17 4- 0. I 1
Change (°C) -0.99 -0.95 - 1.49 - 1.25 - I. 13
_+ 0.17 _+ 0.14 4- 0.10* __ 0.17 4- 0.07**
The hypothermicresponseto apomorphine 1mg/kgi.p. was determined24 hr after the final injectionof morphine. Rectal temperature was measured before and 30 rain after the apomorphine injection. Changes were expressed as the differences of temperature betweenbefore and after the injection. *P < 0.05, comparedwith that of the salinegroup. **P < 0.01, compared with that of the morphinegroup. GP 26/5--M
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Hack-SeangKimet al. 150 w o ¢0
c -•
loo
50
l
0
0<> OO 0<> OO (X) 0<>
Fig. 2. Effect of GTS on the enhanced ambulatory activity of apomorphine in mice treated with morphine for 7 days. The ambulatory activity of apomorphine 4 mg/kg was determined 24 hr after the final injection of morphine 10 mg/kg. Mice were first allowed to preambulate for 10 min and then tested for 20 min. After an interval of 30 min, mice were injected i.p. with apomorphine 4 mg/kg, and then 10 min preambulatory period and 20 rain test period was repeated. Data were expressed as the difference of the activity scores between before and after the injection. ~ 4~P < 0.01, compared with that of the saline group. *P < 0.05, compared with that of the morphine group. IS]:Saline; t~: GTS 200 mg/kg; I~: morphine 10 mg/kg; I'x-]: morphine 10 mg/kg + GTS 100 mg/kg; and II: morphine 10 mg/kg + GTS 200 mg/kg.
to the ambulatory accelerating effects of morphine and methamphetamine, and independently of these, the development of dopamine receptor supersensitivity induced by morphine (Kim et al., 1990; Tokuyama et al., 1992). However, in the present experiment, GTS separated from ginseng extract also inhibited the development of both phenomena induced by morphine. So, it is presumed that GTS is an active compound fraction of ginseng extract on the development of reverse tolerance to the ambulation accelerating effect of morphine and the dopamine receptor supersensitivity. The present experiments show that morphine increases the ambulatory activities of mice and those effects are progressively enhanced by the repeated administration of morphine indicating the development of reverse tolerance. These results are identical with those reported previously (Kuribara and Tadokoro, 1989; McKim, 1986; Shuster, 1975). The phenomenon of reverse tolerance is a model for studying the psychotoxicity of dependence-liable drugs (Allen and Young, 1978; Robinson and Becker, 1986). The effects of GTS on the phenomenon in mice suggest that GTS may be useful in the therapy of the adverse actions of morphine. The effects of morphine on dopamine systems have been reviewed by Wood and Altar (1988). Morphine increased dopamine levels in nucleus accumbens and rat striata DOPAC concentrations increase consistently after morphine administration, indicating the increase of dopamine turnover (Porrino et al., 1988). In association with these facts, it has been presumed that the behavioral sensitization produced by the chronic administration of morphine is accompanied by the change of dopa-
minergic neuronal activity. In addition, it has been demonstrated that such sensitization is blocked by neuroleptics (Beninger and Hahn, 1983; Colpaert et al., 1978; Gawin and Kleber, 1984; Giannini et al., 1986). Therefore, the enhanced responses to apomorphine, a direct-acting dopamine receptor agonist, should be enhanced since the postsynaptic dopamine receptor supersensivity develops after repeated administration of morphine (Bhargava, 1980; Ritzmann et al., 1979). In the present experiments, the enhancements of the hypothermic response to and ambulatory activity by apomorphine were also confirmed in groups treated with morphine, in comparison with that of control group suggesting the development of dopamine receptor supersensitivity. GTS inhibited the development of reverse tolerance to ambulation-accelerating effect of morphine. GTS also inhibited the development of dopamine receptor supersensitivity by the chronic treatment of morphine. In support of this, Kim et al. (1990) demonstrated that standardized ginseng extract inhibited the development of reverse tolerance to the locomotor accelerating activity of morphine and the development of morphine-induced dopamine receptor supersensitivity. And Tokyuama et al. (1992) also demonstrated that standardized ginseng extract suppressed the development of reverse tolerance to the ambulation-accelerating effect of methamphetamine, and presumed that the inhibitory effect of ginseng extract on methamphetamine induced reverse tolerance was related to the recovery of dysfunction in dopaminergic system. However, the possible mechanisms underlying the inhibitory effect of GTS on the development of reverse tolerance and dopamine
Morphine and ginseng receptor supersensitivity to morphine remains unclear. Morphine binds to #-opioid receptors which exist on presynaptic dopaminergic structures (Ritzmann and Bhargava, 1979). Therefore, it is suggested that the inhibitory effects of GTS on the development of reverse tolerance and dopamine receptor supersensitivity to morphine might be associated with the interruption of chronic morphine action at the presynaptic dopamine receptors since morphine action is indirect on dopamine receptors as Puri (1973) and Bhargava (1980) suggested. In conclusion, repeated intermittent administration of morphine developed reverse tolerance to the ambulation-accelerating effect of morphine as well as dopamine receptor supersensitivity. Administration of GTS as an active component fraction of ginseng extract inhibited the development of reverse tolerance to the ambulation-accelerating effect of morphine, and dopamine receptor supersensitivity. It is tempting to speculate that the development of reverse tolerance to the ambulation-accelerating effect of morphine may be closely associated with the development of dopamine receptor supersensitivity since both phenomena were blocked by GTS. The present results have demonstrated that GTS may be useful in the prevention and therapy of the adverse action of morphine.
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produced environment-specificconditioning. Science 220, 1304-1306. Bhargava H. N. (1980) Cyclo (Lcu-Gly) inhibits the development of morphine induced analgesic tolerance and dopamine receptor supersensitivity in rat. Life Sci. 27, 117-123. Bhargava H. N. and Ramarao P. (1991) The effect of panax ginsengon the development of tolerance to the pharmacological actions of morphine in rats. Gen. Pharmae. 22, 521-525.
Colpaert F. C., Niemegcers C. J. E. and Janssen P. A. J. (1978) Neurolcptic interference with the cocaine cue: internal stimulus control of behavior and psychosis. Psychopharmacology 58, 247-255. Dwoskin L. P., Peris J., Yasuda R. P., Philpott K. and Zahnister N. R. (1988) Repeated cocaine administration results in supersensitivity of strial D-2 dopamine autoreceptors to pergolite. Life Sci. 42, 255-262. Gawin F. M. and Kleber D. H. (1984) Cocaine abuse treatment: open pilot trial with desipramine and lithium carbonate. Archs. gen. Psychiat. 11, 903-904. Giannini A. J., Malone D. H. and Giannini M. C. (1986) Treatment of depression in chronic cocaine and phencyclidine abuse with desipramine. J. din. Pharmac. 26, 211-214. Hirabayashi M. and Alam M. R. (1981) Enhancing effect of methamphetamine on ambulatory activity produced by repeated administration in mice. Pharmac. Biochem. Behav. 15, 925-932. Katu T., Miyata T., Urono T., Sako I. and Kinoshita A. (1975) Chemico-pharmacologicalstudies on saponins of panax ginseng C. A. Mayer. Arzneim. Forsch. (Drug Res.) 25, 343-347. Kilbey M. M. and Ellinwood E. H. (1977) Reverse tolerance to stimulant-induced abnormal behavior. Life Sci. 20, 1063-1076. Kim H. S., Jang C. G. and Lee M. K. (1990) Antinarcotic effects of the standardized ginseng extract Gl15 on morphine. Planta Medica 56, 158-163. SUMMARY Kim Y. C., Lee J. H., Kim M. S. and Lee N. G. (1985) Effect on the saponin fraction of panax ginseng on Daily repeated administration of morphine catecholamines in mouse brain. Archs. Pharmac. Res. 8, (10mg/kg, over a 7 day period) developed reverse 45-49. tolerance to the ambulation-accelerating effect of Kuribara H. and Tadokoro S. (1989) Reverse tolerance to ambulation-increasing effects of MAP and MOR in 6 morphine. Intraperitoneal administration of ginseng mouse strains. Jpn. J. Pharmac. 49, 197-203. total saponin (100 and 200mg/kg) prior to and Mansour A., Khachaturian H., Lewis M. E., Akil H. and Watson S. J. (1987) Autoradiographic differentiation of during chronic administration of morphine inhibited mu, delta, and kappa opioid receptors in the rat forebrain the development of reverse tolerance. Dopamine and midbrain. J. Neurosci. 7, 2445-2464. receptor supersensitivity was also developed in McKim W. A. (1986) Psychomotor stimulants and antidepressants. In Drugs and Behavior: An Introduction to reverse tolerant mice with the same treatment of Behavioral Pharmacology, pp. 161-185. Prentice-Hall, morphine. GTS also prevented the development of Englewood Cliffs, N.J. dopamine receptor supersensitivity induced by the Namba T., Yoshizaki T., Tominori K., Kobashi K., Mitsui K. and Hasse J. (1974) Fundamental studies on the chronic administration of morphine. These results evaluation of the crude drugs (l). Planta Medica 32, suggest that GTS may be useful for the prevention 588-594. and therapy of the adverse action of morphine. Petkov V. W. (1959) Pharmacological investigation of the drug Panax ginseng C. ,4. Meyer. Arzneim. Forsch. (Drug Res.). 9, 305-311. Acknowledgements--This study was supported by a grant from the New Drug Development, Ministry of Health and Porrino L. J., Domer F. R., Crane A. M. and Sokoloff L. (1988) Selective alternations in cerebral metabolism Social Affairs, Republic of Korea (1993). The authors thank Ginseng and Tobacco Research Institute for the supply of within the mesocorticolimbic dopaminergic system proginseng total saponin. duced by acute cocaine administration of rats. Neuropsychopharmacology 1, 109-118. Puri S. K. and Lal H. (1973) Effect of dopaminergic stimulation or blockade on morphine-withdrawal aggresREFERENCES sion. Psychopharmacologia 32, 113-120. Allen R. M. and Young S. J. (1978) Phencyclidine-induced Ritzmann R. F., Bhargava H. N. and Flexner, L. B. (1979) psychosis. Am. J. Psychiat. 135, 1081-1084. Blockade of narcotic-induced dopamine receptor superBeninger R. J. and Hahn B. U (1983) Pimozide blocks sensitivity by cyclo (Leu-Gly). Proc. nam. Acad. Sci. establishment but not expression of amphetamineU.S.A. 76, 5997-5998.
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Robinson T. and Becker J. (1986) Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis. Brain Res. Rev. I1, 157-198. Saito H., Morita M. and Takagi K. (1973) Pharmacological studies of panax ginseng leaves. Jpn. J. Pharmacol. 23, 43-56. Shuster L., Webster G. W. and Yu G. (1975) Increased running response to morphine in morphine-pretreated mice. J. Pharmac. Exp. Ther. 192, 64-72. Takagi K., Saito H. and Nabata H. (1972) Pharmacological studies of panax ginseng root: estimation of pharmacological actions of panax ginseng root. Jpn. J. Pharmac. 22, 245-259.
Taylor D., Ho B. T. and Fager J. D. (1979) Increased dopamine receptor binding in rat by repeated cocaine injections. Commun. Psycho. Pharmac. 3, 137-142. Tokuyama S., Oh K. W., Kim H. S., Takahashi M. and Kaneto H. (1992) Blockade by ginseng extract of the development of reverse tolerance to the ambulationaccelerating effect of methamphetamine in mice. Jpn. J. Pharmac. 59, 423-425. Tsang D., Yeung H. W., Tso W. W. and Peck H. (1985) Ginseng saponins influence on neurotransmitter in rat brain synaptosomes. Planta Medica 3, 221-224. Wood P. L. and Altar C. A. (1988) Dopamine release in vivo from nigrostriatal mesolimbic and mesocortical neurons utility of 3-methoxytryramine measurements. Pharmac. Rev. 40, 163-187.
0306-3623(94)00267-3
Gen. Pharmac.Vol. 26, No. 5, pp. 1071-1076, 1995 Copyright© 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/95 $9.50+ 0.00
Inhibition by Ginseng Total Saponin of the Development of Morphine Reverse Tolerance and Dopamine Receptor Supersensitivity in Mice HACK-Seang KIM,* JIN-Goo KANG and KI-Wan OH Department of Pharmacology, College of Pharmacy, Chungbuk National University, Cheong/u 360-763, Korea [Fax: (431) 68-27321 (Received 13 September 1994)
Abstraet--l. Ginseng total saponin (GTS), 200mg/kg i.p. 3hr prior to morphine, inhibited the development of reverse tolerance to the ambulatory-accelerating effect of morphine. 2. GTS, 200 mg/kg, also prevented the development of dopamine receptor supersensitivity induced by the chronic administration of morphine, 10 mg/kg a day for 7 days. 3. These results suggest that GTS may be useful for the prevention and therapy of the adverse action of morphine.
Key Words: Ginseng total saponin (GTS), reverse tolerance, morphine, supersensitivity
INTRODUCTION The pharmacological effects of ginseng on various organs have been reviewed (Katu et al., 1975; Saito et al., 1973). Petkov (1959) demonstrated that ginseng extract had a variety of effects on the activity of the central nervous system (CNS). Ginseng not only stimulates in low dose but also inhibits the CNS (Takagi et al., 1972). It also acts as a modulator of neurotransmission in the brain (Kim et al., 1985; Tsang et al., 1985). Dopaminergic and noradrenergic neurons in the CNS play important roles in the behavioral effects of drugs (Dwoskin et al., 1988; Porrino et al., 1988; Puri and Lal, 1973; Taylor et al., 1979; Wood and Altar, 1988). The repeated intermittent administration of CNS stimulants to animals produces progressive changes in behavior, a phenomenon known as behavioral sensitization (reverse tolerance) (Hirabayashi and Alam, 1981; Kilbey and Ellinwood, 1977; Kuribara and Tadokoro, 1989; Mansour et aL, 1987; Shuster et al., 1975). It is thought that this sensitization represents an animal model of drug*To whom all correspondence should be addressed.
induced psychosis (Allen and Young, 1978; Robinson and Becker, 1986). It is also reported that rats sensitized to morphine show an enhanced response to apomorphine, a direct dopamine receptor agonist, suggesting the development of dopamine receptor supersensitivity (Bhargava, 1980; Ritzmann et al., 1979). Behavioral sensitization after the repeated administration of morphine has been demonstrated to be attributable to the dopaminergic hyperfunction in the CNS (Kilbey and Ellinwood, 1977; Puri and Lal, 1973; Robinson and Becker, 1986; Taylor et al., 1979). On the other hand, Bhargava and Ramarao (1991) reported that ginseng extract had an inhibitory activity on the development of tolerance to the pharmacological actions of morphine. In addition, Tokuyama et al. (1992) have shown that the standardized ginseng extract prevents the development of reverse tolerance to the ambulation-accelerating effect of methamphetamine. Therefore, the present experiments were undertaken to determine the effects of GTS, as an active component fraction of ginseng extract, on the development of reverse tolerance to the ambulationaccelerating effect of morphine in mice. The effects
1071
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Hack-Seang Kim et al.
of GTS on the dopamine receptor supersensitivity induced by morphine were also examined. MATERIALS AND M E T H O D S
Animals and drugs ICR male mice weighing 20-30 g in a group of 10-12, were used in all experiments. They were housed in the acrylic fiber cage in a controlled room (22 ___I°C), and were kept on unlimited supply of the solid diet and tap water. The drugs used were morphine hydrochloride (Dae-Won Pharm. Co., Korea), apomorphine hydrochloride (Sigma, U.S.A.) and GTS [characterized saponins mixture containing at least 10 glycosides Rb 2 (18.26%), Rb2 (9.07), Rc (9.65%), Rd (8.24%), Re (9.28%), Rf (3.48%), Rgl (6.42%), Rg 2 (3.62%), Rg 3 (4.70%), Ro (3.82%), Ra (2.91%) and minor ginsenosides and components (20.55%) from panax ginseng, extracted and purified by Namba et al.'s method (Namba et al., 1974) and supplied by Korea Ginseng and Tobacco Research Institute]. Except for apomorphine, the drugs were dissolved in physiological saline. Apomorphine was dissolved in saline containing 0.1% ascorbic acid, just prior to the experiment. Morphine was administered to mice subcutaneously (s.c.), and GTS and apomorphine were administered to mice intraperitoneally (i.p.).
Measurement of the development of reverse tolerance to the ambulation-accelerating effect As the development of "reverse tolerance" is subject to such variations as drug dosage, route of administration and duration of treatment, for each endpoint investigated, parameters were selected empirically to produce a significant degree of reverse tolerance within a period of 7 days or less. For example, the rate of development of reverse tolerance is generally dependent on the amount of dosage, therefore, the daily drug dosage was designed to produce a significant change in endpoint within the limited duration of the study. Furthermore, in mouse, reverse tolerance to morphine develops more expeditiously to s.c. than i.p. administration, hence the former route of administration was used to produce reverse tolerance in these experiments. The ambulatory activity of mice was measured by the tilting-type ambulometer (AMB-10, O'Hara & Co., Japan). Each mouse was placed in activity cages of 20 cm in diameter and 18 cm in height. Drug administration was carried out after an adaptation period of 10rain. Daily ambulatory activity was measured for 3 hr after morphine administration. Morphine 10 mg/kg was administered once per day for 7 days. The development of reverse
tolerance was evidenced by the enhanced ambulationaccelerating activity with the repeated administration of morphine. When the combined effect of morphine and GTS was investigated at various time intervals, the consistent and reliable inhibitory effects of GTS were observed in mice pretreated with GTS 3 hr prior to morphine administration. Therefore, mice were pretreated with GTS 100 or 200 mg/kg 3 hr prior to morphine injection.
Measurement of the development of dopamine receptor supersensitivity The development of dopamine receptor supersensitivty was determined by measuring the enhanced hypothermic response to and the enhanced ambulatory activity of apomorphine (a dopaminergic receptor agonist) in mice treated with morphine. Additional groups of mice with the same chronic morphine and GTS treatment, as in the measurement of the development of reverse tolerance in the previous test, were used to determine the effects of these treatments on the hypothermic response to apomorphine. The hypothermic response to an i.p. injection of apomorphine 1 mg/kg (Ritzmann, 1979), a dosage sufficient to produce hypothermia, was determined 24 hr after the final injection of morphine. Body temperature was determined by using a rectal probe (inserted 2.5 cm into the rectum) and a telethermometer. The measurements were made just prior to and 30 min after apomorphine injection. Data were expressed as the difference between the measurements before and after the injection of apomorphine. Groups of mice with the same chronic morphine and GTS treatment as in the previous test, were used to determine the effects of these treatments on the apomorphine induced ambulation-accelerating activity. The ambulationaccelerating activity of apomorphine was measured 24 hr after the final injection of morphine. Mice were first allowed to preambulate for 10min followed by a 20min test period. After an interval of 30min, mice were given apomorphine 4mg/kg, a dosage which produced significant increase of ambulatory activity (Bhargava, 1980). Immediately after administration, the 10min preambulation period and 20 min test period were repeated. Data were expressed as the difference between the test activity counts before and after the injection of apomorphine.
Statistics The data expressed were as mean _ SE. The significant differences were first analyzed by various (ANOVA). In case of significant variation, the individual values were compared by Student's t-test.
Morphine and ginseng 3000
H ~ 0
O# v H
M
0
2000
-/°
L
?
, "/T &/.
1000
a.._.~ [] •
..
..
1073
tration of the drugs with 2600 counts on day 6, compared with 1370 counts on day 1. GTS given 3 hr before morphine injection, dose-dependently suppressed the development of reverse tolerance to morphine. The activity count on day 6, was approx. 1400, about 1200 less than that of the morphine pretreated group in mice pretreated with GTS 200 mg/kg, (Fig. 1). These results suggest that GTS inhibits the development of reverse tolerance to the ambulation-accelerating effects of morphine.
Inhibitory effects of GTS on the development of dopamine receptor supersensitivity 1
2
3
4
5
8
?
Fig. I. Effect of GTS on the development of reverse tolerance to the ambulation-accelerating effect of morphine. Morphine I0 mg/kg (s.c.) was administered to mice once a day for 7 days. GTS I00 or 200 mg/kg (i.p.) was administered to mice 3 hr prior to the injection of morphine. The ambulatory activity was measured for 1 hr after morphine administration. 4fP < 0.05; # # P < 0.01, compared with the count on day I. *P <0.05; **P <0.01, compared with that of the morphine group. © ©: Saline; • O: morphine 10 mg/kg; A A: GTS 100 mg/kg; V V: GTS 200 mg/kg; O O: morphine 10mg/kg+GTS 100mg/kg; and [] I:]: morphine l0 mg/kg + GTS 200 mg/kg.
RESULTS
Inhibitory effects of GTS on the development of morphine induced reverse tolerance to the ambulationaccelerating effect Figure 1 shows the mean overall ambulatory activity after the repeated administration of morphine (10 mg/kg). The ambulation-accelerating activities of morphine were enhanced by repeated treatment, i.e. reverse tolerance was developed to the ambulation-accelerating effects of morphine. Single or chronic treatment with GTS or saline alone did not influence the spontaneous motor activity of mice. Morphine induced ambulation-accelerating activity was markedly enhanced by the repeated adminis-
The hypothermic response to apomorphine I mg/kg was enhanced in mice treated with morphine, in comparison to the saline treatment group. Morphine treated mice receiving chronic injection of GTS 100 or 200mg/kg 3 hr prior to the injection of morphine did not exhibit an enhanced hypothermic response to apomorphine (Table 1). Single or chronic treatment with GTS or saline alone did not influence the hypothermic response of mice. The enhanced hypothermic response of apomorphine in chronic morphine treated mice was inhibited by GTS 200 mg/kg pretreatment 3 hr prior to the injection of morphine (Table 1). Apomorphine at a dose of 4 mg/kg produced a significant increase of ambulatory activity in chronic treated mice, compared with the increase observed in saline treated mice. The enhanced ambulatory activity of apomorphine in chronic morphine treated mice was inhibited by GTS 200 mg/kg pretreatment 3 hr prior to the injection of morphine (Fig. 2). These results make it evident that chronic administration of morphine develops dopamine receptor supersensitivity, and that GTS blocks the development of dopamine receptor supersensitivity. DISCUSSION It has been reported that the standardized ginseng extract inhibited the development of reverse tolerance
Table I. Effect o f G T S on the enhanced hypothermic response to apomorphine in mice treated with m o r p h i n e
Body temperature(°C) Group Saline G T S 200 Saline + m o r p h i n e G T S 100 + morphine G T S 200 + m o r p h i n e
At 0 rain 38.20 38.23 38.14 38.24 38.06
4- 0.24 +_ 0.22 4- 0.10 -4- 0.10 4- 0.13
At 30 min 37.21 37.28 36.65 36.99 36.93
_ 0.10 4- 0.17 _ 0.11 __ 0.17 4- 0. I 1
Change (°C) -0.99 -0.95 - 1.49 - 1.25 - I. 13
_+ 0.17 _+ 0.14 4- 0.10* __ 0.17 4- 0.07**
The hypothermicresponseto apomorphine 1mg/kgi.p. was determined24 hr after the final injectionof morphine. Rectal temperature was measured before and 30 rain after the apomorphine injection. Changes were expressed as the differences of temperature betweenbefore and after the injection. *P < 0.05, comparedwith that of the salinegroup. **P < 0.01, compared with that of the morphinegroup. GP 26/5--M
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Hack-SeangKimet al. 150 w o ¢0
c -•
loo
50
l
0
0<> OO 0<> OO (X) 0<>
Fig. 2. Effect of GTS on the enhanced ambulatory activity of apomorphine in mice treated with morphine for 7 days. The ambulatory activity of apomorphine 4 mg/kg was determined 24 hr after the final injection of morphine 10 mg/kg. Mice were first allowed to preambulate for 10 min and then tested for 20 min. After an interval of 30 min, mice were injected i.p. with apomorphine 4 mg/kg, and then 10 min preambulatory period and 20 rain test period was repeated. Data were expressed as the difference of the activity scores between before and after the injection. ~ 4~P < 0.01, compared with that of the saline group. *P < 0.05, compared with that of the morphine group. IS]:Saline; t~: GTS 200 mg/kg; I~: morphine 10 mg/kg; I'x-]: morphine 10 mg/kg + GTS 100 mg/kg; and II: morphine 10 mg/kg + GTS 200 mg/kg.
to the ambulatory accelerating effects of morphine and methamphetamine, and independently of these, the development of dopamine receptor supersensitivity induced by morphine (Kim et al., 1990; Tokuyama et al., 1992). However, in the present experiment, GTS separated from ginseng extract also inhibited the development of both phenomena induced by morphine. So, it is presumed that GTS is an active compound fraction of ginseng extract on the development of reverse tolerance to the ambulation accelerating effect of morphine and the dopamine receptor supersensitivity. The present experiments show that morphine increases the ambulatory activities of mice and those effects are progressively enhanced by the repeated administration of morphine indicating the development of reverse tolerance. These results are identical with those reported previously (Kuribara and Tadokoro, 1989; McKim, 1986; Shuster, 1975). The phenomenon of reverse tolerance is a model for studying the psychotoxicity of dependence-liable drugs (Allen and Young, 1978; Robinson and Becker, 1986). The effects of GTS on the phenomenon in mice suggest that GTS may be useful in the therapy of the adverse actions of morphine. The effects of morphine on dopamine systems have been reviewed by Wood and Altar (1988). Morphine increased dopamine levels in nucleus accumbens and rat striata DOPAC concentrations increase consistently after morphine administration, indicating the increase of dopamine turnover (Porrino et al., 1988). In association with these facts, it has been presumed that the behavioral sensitization produced by the chronic administration of morphine is accompanied by the change of dopa-
minergic neuronal activity. In addition, it has been demonstrated that such sensitization is blocked by neuroleptics (Beninger and Hahn, 1983; Colpaert et al., 1978; Gawin and Kleber, 1984; Giannini et al., 1986). Therefore, the enhanced responses to apomorphine, a direct-acting dopamine receptor agonist, should be enhanced since the postsynaptic dopamine receptor supersensivity develops after repeated administration of morphine (Bhargava, 1980; Ritzmann et al., 1979). In the present experiments, the enhancements of the hypothermic response to and ambulatory activity by apomorphine were also confirmed in groups treated with morphine, in comparison with that of control group suggesting the development of dopamine receptor supersensitivity. GTS inhibited the development of reverse tolerance to ambulation-accelerating effect of morphine. GTS also inhibited the development of dopamine receptor supersensitivity by the chronic treatment of morphine. In support of this, Kim et al. (1990) demonstrated that standardized ginseng extract inhibited the development of reverse tolerance to the locomotor accelerating activity of morphine and the development of morphine-induced dopamine receptor supersensitivity. And Tokyuama et al. (1992) also demonstrated that standardized ginseng extract suppressed the development of reverse tolerance to the ambulation-accelerating effect of methamphetamine, and presumed that the inhibitory effect of ginseng extract on methamphetamine induced reverse tolerance was related to the recovery of dysfunction in dopaminergic system. However, the possible mechanisms underlying the inhibitory effect of GTS on the development of reverse tolerance and dopamine
Morphine and ginseng receptor supersensitivity to morphine remains unclear. Morphine binds to #-opioid receptors which exist on presynaptic dopaminergic structures (Ritzmann and Bhargava, 1979). Therefore, it is suggested that the inhibitory effects of GTS on the development of reverse tolerance and dopamine receptor supersensitivity to morphine might be associated with the interruption of chronic morphine action at the presynaptic dopamine receptors since morphine action is indirect on dopamine receptors as Puri (1973) and Bhargava (1980) suggested. In conclusion, repeated intermittent administration of morphine developed reverse tolerance to the ambulation-accelerating effect of morphine as well as dopamine receptor supersensitivity. Administration of GTS as an active component fraction of ginseng extract inhibited the development of reverse tolerance to the ambulation-accelerating effect of morphine, and dopamine receptor supersensitivity. It is tempting to speculate that the development of reverse tolerance to the ambulation-accelerating effect of morphine may be closely associated with the development of dopamine receptor supersensitivity since both phenomena were blocked by GTS. The present results have demonstrated that GTS may be useful in the prevention and therapy of the adverse action of morphine.
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