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Role of benzodiazepine–GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice
Life Sciences 70 (2002) 1727 – 1740
Role of benzodiazepine–GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice Kumar V.S. Nemmani, P. Ramarao* Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Phase - X, S.A.S. Nagar - 160 062 (Pb), India Received 27 June 2001; accepted 1 October 2001
Abstract In the present study, the role of benzodiazepine – GABAA receptor complex in the attenuation of U50,488H (U50), a selective kappa opioid agonist-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin (GTS) was investigated using the mice tail –flick test. The intraperitoneal (i.p.) treatment of GTS (100 and 200 mg/kg) and diazepam (0.1 – 1 mg/kg) dosedependently attenuated the U50 (40 mg/kg, i.p.)-induced analgesia. GTS (0.001 – 10 mg/ml) did not alter binding of [3H]naloxone to mice whole brain membrane. The attenuation effect of GTS (100 mg/ kg) and diazepam (0.5 mg/kg) on U50-induced analgesia was blocked by flumazenil (0.1 mg/kg, i.p.), a benzodiazepine receptor antagonist, and picrotoxin (1 mg/kg, i.p.), a GABAA-gated chloride channel blocker. However, bicuculline (1 mg/kg, i.p.), a GABAA receptor antagonist blocked the attenuation effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on U50-induced analgesia. Chronic treatment (day 4 – day 6) of GTS (50 –200 mg/kg) and diazepam (0.1 – 1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) on chronic treatment blocked the inhibitory effect of GTS (100 mg/kg) and diazepam (0.5 mg/kg) on tolerance to U50-induced analgesia. On the other hand, chronic treatment of bicuculline (1 mg/kg) blocked the inhibitory effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on tolerance to U50induced analgesia. In conclusion, the findings suggest that GTS attenuates U50-induced analgesia and
* Corresponding author. Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Phase - X, S.A.S. Nagar - 160 062 (Pb), India. Tel.: +91-172-214-683x2043; fax: +91-172-214-692. E-mail address: [email protected] (P. Ramarao). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 4 9 6 - 0
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inhibits tolerance to its analgesia and this action involves benzodiazepine receptors and GABAA-gated chloride channels. D 2002 Elsevier Science Inc. All rights reserved. Keywords: ginseng total saponin; diazepam; kappa opioid agonist; analgesia; tolerance; benzodiazepine – GABAA receptor complex
Introduction The use of Panax ginseng, C. A. Meyer (Aralaciaceae) root as a folklore medicine in the far eastern countries goes back to thousands of years. Many studies indicate that ginseng saponins are the major active constituents of Panax ginseng [1]. Ginseng total saponin (GTS), a mixture of total saponins of Panax ginseng and some of its saponins have been demonstrated to attenuate morphine, a mu opioid- and U-50,488H (U50), a selective kappa opioid-induced analgesia in the mice tail–flick test [2,3]. Further, GTS and ginseng extracts have been reported to inhibit tolerance to morphine- induced analgesia in rodents [4–6]. Recently, we reported that GTS, panaxadiol and panaxatriol inhibit tolerance to U50-induced analgesia in mice [7]. However, the mechanism(s) by which GTS is able to attenuate opioid-induced analgesia and inhibit tolerance to its analgesia are not known. Several studies indicate that GABAergic mechanisms play a role in the modulation of muand kappa opioid-induced analgesia [8–11]. Diazepam, a benzodiazepine receptor agonist is well known to produce many of its effects through enhancement of GABAergic neurotransmission [12]. Further, the attenuation effect of diazepam on mu-and kappa opioidinduced analgesia was blocked by flumazenil, a benzodiazepine receptor antagonist and picrotoxin, a GABAA receptor-gated chloride channel blocker suggesting the involvement of benzodiazepine – GABAAergic mechanisms in attenuation of opioid-induced analgesia [11,13]. In addition, recent studies indicated that diazepam and midazolam inhibit tolerance to morphine-induced analgesia [14–17]. Ginseng extracts block the uptake of GABA into rat brain synaptosomes [18]. Moreover, GTS and some of its saponins are reported to alter the binding of GABAA [19]. In addition, ginseng is demonstrated to have diazepam like activity [20]. Since GTS has diazepam like activity, block the uptake of GABA and alter the binding of GABAA ligands, we hypothesise that GTS possibly attenuates U50-induced analgesia and inhibits tolerance to its analgesia via benzodizepine–GABAAergic mechanisms. Hence, in the present studies the possible role of benzodiazepine –GABAAergic mechanisms in attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS are investigated using the selective antagonists of benzodiazepine–GABAA receptor complex in the mice tail–flick test. Methods Animals Swiss male mice (Central Animal Facility, NIPER, India) weighing 20–26 g were housed five to a cage in a room with controlled temperature (22 ± 2 C), humidity (50 ± 10%) and
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light (06.00–18.00 h). Food and water were made available ad libitum. All experiments were performed between 10.00 and 16.00 h to minimize diurnal variations. Measurement of analgesic response by tail–flick test in mice The analgesic response was determined as described previously by modified tail–flick test [7]. Briefly, the tail flick latencies to thermal stimulation (radiant heat) were determined at 0, 30, 60, 90, 120 and 180 min after administration of U50. The basal latencies were found to 5–7 sec. A cutoff time of 20 sec was followed to prevent any injury to the tail. The percent maximum possible effect (%MPE) was calculated using the formula % MPE ¼ ðPost latency Basal latencyÞ=ðCutoff latency Basal latencyÞ 100
ð1Þ
A graph is plotted as Mean % MPE ± S.E.M. vs time. The area under curve (AUC 0 – 180 min.) of %Mean % MPE ± S.E.M. vs time plot was calculated for different groups and the data was subjected to statistical analysis. Induction and assessment of tolerance to U50-induced analgesia in mice Mice were rendered tolerant to U50 by administration of U50 (40 mg/kg) daily in the morning and evening for six days [7]. Mice serving as control were administered similarly with vehicle. On day 7 analgesic response was assessed as described above in separate groups of mice challenging with U50 (40 mg/kg). The AUC 0 – 180 min was calculated for each mouse and subjected to statistical analysis. Determination of displacement of [3H]naloxone (4nM) binding to mice whole brain by GTS Binding studies were performed with slight modification of the method described by Yoburn et al [21]. Mice were sacrificed and whole brains were removed, weighed and homogenised in 20 volumes of ice-cold 50 mM Tris buffer (PH 7.6). The homogenates were then centrifuged (49000 g, 4 C, and 15 min) and the pellet was resuspended in buffer and incubated (30 min, 25 C). Homogenate was again centrifuged and supernatant was removed and suspended in 20 volumes of Tris buffer. An aliquot of brain homogenate containing 250– 300 mg of protein was assayed in triplicate containing 4 nM of [3H]naloxone. The amount of ligand displaced by GTS was determined at the concentration range of 0.001–10 mg/ml. Similarly, a parallel study was conducted in triplicate with naloxone (0.001 nM–1 mM). Homogenate was incubated for 90 min at 25 C. To the homogenate ice-cold buffer was added and then filtered using Brandel filtration unit with GF/B whatman filter paper. The tubes were washed with buffer, filter papers were then transferred to scintillation vials and cocktail was added. After overnight saturation, the samples were counted using Wallac 1407 b-counter (Efficiency 49%).
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Drugs The substances used in the study were U50 (Pharmacia and Upjohn Company, Kalamazoo, USA), GTS (Korea Ginseng and Tobacco Research Institute, Taejon, Korea) which is mixture of ginsenosides and the ratios of various ginsenosides were Rb1 (18.26%), Rb2 (9.07%), Rc (9.65%), Rd (8.24%), Re (9.28%), Rf (3.48%), Rg1 (6.42%), Rg2 (3.63%), Rg1 (4.70%), R0 (3.82%), Ra (2.91%) and other minor ginsenosides, diazepam (Ranbaxy Laboratories, Dewas, India), flumazenil (F.Hoffman -La Roche AG, Basel, Switzerland), [N-allyl2,3-3H]naloxone (Amersham Life science, England), naloxone (Sigma Chemical, St. Louis, USA), picrotoxin (Sigma Chemical, St. Louis, USA) and bicuculline (Sigma Chemical, St. Louis, USA). U50 and GTS were dissolved in distilled water. Diazepam and flumazenil were dissolved in 2% DMSO-water. Picrotoxin was dissolved in 2% ethanol–water whereas, bicuculline was initially dissolved in few drops of 1N HCl and diluted with distilled water. All substances were prepared freshly and administered i.p. in a volume of 10 ml/kg of body weight. Treatment schedule In the acute study, GTS and diazepam were administered 4 hr and 30 min prior to U50 administration, respectively. Whereas, flumazenil, picrotoxin and bicuculline were administered 1 hr prior to U50 administration. In tolerance studies, GTS and diazepam were administered chronically (day 4–day 6) once daily 4 hr and 30 min prior to evening dose of U50, respectively. Similarly, flumazenil, picrotoxin and bicuculline were administered chronically (day 4–day 6) 1 hr prior to evening dose of U50. Statistical analysis The data was analyzed by one-way analysis of variance (one-way ANOVA) followed by a post-hoc multiple comparison test (Scheffe’s S–test). A value of p< 0.05 was considered to be significant.
Results Effect of GTS and diazepam on U50-induced analgesia in mice The effect of GTS (50, 100 and 200 mg/kg) and diazepam (0.1, 0.5 and 1 mg/kg) on timecourse of action and AUC 0 – 180 min of U50-induced analgesia in mice tail–flick test is shown in Fig. 1. U50 (40 mg/kg, i.p.) produced analgesia with peak maximal effect at 30 min after the administration. GTS (50–200 mg/kg) and diazepam (0.1–1 mg/kg) per se did not produce analgesia (data not shown). GTS (100 and 200 mg/kg) attenuated the peak maximal response of U50-induced analgesia (Fig. 1A). The % decrease in analgesic response to U50 (expressed as AUC 0 – 180 min) by GTS (100 and 200 mg/kg) were 45.5 ± 3.6 and 58.6 ± 3.9,
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Fig. 1. Effect of GTS (50, 100 and 200 mg/kg, i.p.) and diazepam (0.1, 0.5 and 1 mg/kg, i.p.) on time-course of action and AUC 0 – 180min of U50 (40 mg/kg, i.p.)-induced analgesia in the mice tail – flick test. GTS (A) and diazepam (B) were treated at 4 hr and 30 min prior to U50 administration, respectively. # P< 0.05 vs Vehicle.
respectively (Fig. 1A). Diazepam (0.1, 0.5 and 1 mg/kg) also dose-dependently attenuated the U50-induced analgesia (Fig. 1B). The % decrease in analgesic response to U50-induced analgesia by diazepam (0.1, 0.5 and 1 mg/kg) were 43.0 ± 3.2, 55.3 ± 6.2 and 80.2 ± 3.9, respectively (Fig. 1B). Effect of flumazenil, picrotoxin and bicuculline on the attenuation of U50-induced analgesia by GTS and diazepam The effect of flumazenil, picrotoxin and bicuculline on attenuation of U50-induced analgesia by GTS and diazepam is shown in Fig. 2. GTS (100 mg/kg) and diazepam
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Fig. 2. Effect of flumazenil (0.1 mg/kg, i.p.), picrotoxin (1 mg/kg, i.p.), and bicuculline (1 mg/kg, i.p.) on attenuation of U50 (40 mg/kg)-induced analgesia by GTS (100 mg/kg, i.p., 4 hr prior) and diazepam (0.5 mg/kg, i.p., 30 min prior) in mice. Flumazenil (A), picrotoxin (B) and bicuculline (C) were treated one hr prior to U50 administration. Unfilled bar indicates control mice; Hatched bars indicate treated mice; All the values are expressed as Mean ± S. E. M., n = 5. * P< 0.05 vs control; # P< 0.05 vs respective GTS or diazepam.
(0.5 mg/kg) attenuated the U50 (40 mg/kg)-induced analgesia. Flumazenil (0.1 mg/kg), picrotoxin (1 mg/kg) and bicuculline (1 mg/kg) per se did not produce analgesia (data not shown) or altered U50-induced analgesia (Fig. 2). However, flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) blocked the attenuation of U50-induced analgesia by GTS and
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Fig. 3. Effect of GTS (0.001 – 10 mg/ml) and naloxone (0.001 nM – 1mM) on binding of 4 nM [3H]naloxone to mice whole brain membrane. All the values are expressed as Mean ± S. E. M., n = 3.
diazepam (Fig 2 A and B). On the other hand, bicuculline (1 mg/kg) blocked the attenuation of U50-induced analgesia by diazepam but not GTS (Fig. 2 C). Table 1 Effect of GTS and diazepam on tolerance to U50-induced analgesia in mice Treatment Group
Analgesic Response to U50 AUC Mean ± S.E.M (n = 5)
GTS Treatment groups Vehicle + Vehicle U50 Treated + Vehicle U50 Treated + GTS (50) U50 Treated + GTS (100) U50 Treated + GTS (200)
2731.2 591.9 1542.5 2662.0 2763.9
± ± ± ± ±
103.6 128.0 * 120.1 # 112.9 # 119.6 #
Diazepam treatment groups Vehicle + Vehicle U50 Treated + Vehicle U50 Treated + Diazepam (0.1) U50 Treated + Diazepam (0.5) U50 Treated + Diazepam (1)
2920.8 501.9 1791.7 2625.4 2721.8
± ± ± ± ±
135.8 94.9 * 200.4# 263.2# 175.2#
0 – 180 min
@ Mice were treated with U50 (40 mg/kg, i.p.) twice daily for six days. GTS (50, 100 and 200 mg/kg, 4 h prior) and diazepam (0.1, 0.5 and 1 mg/kg, 30 min prior) were administered once daily on day 4 - day 6 prior to the evening dose of U50. Analgesic response to U50 (40 mg/kg) was determined on day 7 in mice from all groups. * p < 0.05 vs respective Vehicle + Vehicle group; # p < 0.05 vs respective U50 Treated + Vehicle group.
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Fig. 4. Effect of flumazenil (0.1 mg/kg, i.p.), picrotoxin (1 mg/kg, i.p.), and bicuculline (1 mg/kg, i.p.) on inhibition of tolerance to U50 (40 mg/kg)-induced analgesia by GTS (100 mg/kg, i.p.) and diazepam (0.5 mg/kg, i.p.) in mice. Mice were rendered tolerant to U50 by twice daily administration of U50 (40 mg/kg, i.p.) for six days. GTS and diazepam were treated once daily on day 4 - day 6 at 4hr and 30 min prior to evening dose of U50, respectively. Whereas, flumazenil (A), picrotoxin (B) and bicuculline (C) were treated one hr prior to evening dose of U50 on day 4 - day 6. The analgesic response to U50 (40 mg/kg, i.p.) was determined on day 7. Unfilled bar indicates U50naive and hatched bars indicate U50-tolerant. All the values are expressed as Mean ± S. E. M., n = 5. * P< 0.05 vs U50-naive; # P< 0.05 vs U50-tolerant; @ P< 0.05 vs U50-tolerant + respective GTS or diazepam.
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Effect of GTS on [3H]naloxone binding to mice whole brain The effect of GTS on binding of [3H]naloxone to mice whole brain membrane is shown in Fig. 3. As shown in Fig. 3, GTS did not alter the [3H]naloxone binding. On the other hand, naloxone displaced the [3H]naloxone binding with IC50 of 0.52 nM ± 0.07. Effect of chronic treatment of GTS and diazepam on tolerance to U50-induced analgesia Twice daily administration of U50 (40 mg/kg) for six days resulted in the development of tolerance to U50-induced analgesia. As shown in the Table 1, chronic administration of U50 decreased the analgesic response to U50 by about five folds when compared to that of vehicle administered group. Chronic treatment of GTS (50, 100 and 200 mg/kg) and diazepam (0.1, 0.5 and 1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia (Table 1). Chronic treatment of GTS (50, 100 and 200 mg/kg) to U50-tolerant mice restored the analgesic response to U50 to 56.5 % ± 4.4, 97.5 % ± 4.1 and 101.2 % ± 4.4, respectively. Moreover, chronic administration of diazepam (0.1, 0.5 and 1 mg/kg) to U50-tolerant mice also restored the analgesic response to U50 to 58.4 % ± 8.0, 89.9 % ± 9.0 and 93.2 % ± 6.0, respectively in mice (Table 1). Effect of flumazenil, picrotoxin and bicuculline on the inhibition of tolerance to U50-induced analgesia by GTS and diazepam The effect of flumazenil, picrotoxin and bicuculline on the inhibition of tolerance to U50induced analgesia by GTS and diazepam is shown in Fig. 4. Twice daily administration of U50 (40 mg/kg) for six days resulted in the development of tolerance to U50-induced analgesia (Fig. 4). As shown in the Fig. 4, chronic treatment of GTS (100 mg/kg), and diazepam (0.5 mg/kg) inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/ kg) and picrotoxin (1 mg/kg) on chronic treatment did not alter the tolerance to U50-induced analgesia. However, chronic treatment of flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) blocked the inhibition of tolerance to U50-induced analgesia by GTS and diazepam (Fig. 4 A and B). On other hand, bicuculline per se inhibited the tolerance to U50-induced analgesia. In addition, bicuculline blocked the inhibition of tolerance to U50-induced analgesia by diazepam but not GTS (Fig. 4 C).
Discussion U50, a selective kappa opioid agonist appears to produce analgesia by activation of descending pain inhibitory system in mice tail–flick test [22]. Recent studies indicate that intracerebroventricularly (i.c.v.) and intrathecaly (i.t.) administered GTS and its saponins dose-dependently attenuate kappa opioid-induced analgesia in the mice tail–flick test [2]. Further, i.c.v. administered GTS inhibits tolerance to morphine-induced analgesia [4]. We reported recently that systemic treatment of GTS and its two major fractions viz panaxadiol
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and panaxatriol inhibit tolerance to U50-induced analgesia [7]. However, the mechanism(s) by which GTS exerts its effects on opioid-induced analgesia and its tolerance to analgesia are not known. In an attempt to determine the mechanism(s) of attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS, the effect of antagonists of benzodiazepine– GABAA receptor complex on attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS were investigated in the present study. In consistent with the literature, we found that systemic treatment of GTS dose-dependently attenuates U50induced analgesia [3]. In this study, we also found that systemic treatment of diazepam dosedependently attenuates U50-induced analgesia, and that the effect of diazepam was blocked by flumazenil, picrotoxin and bicuculline. Thus these findings support the idea that enhancement of benzodiazepine–GABAergic transmission negatively modulates the kappa opioid induced analgesia [11,13]. In this study, we also found that the attenuation effect of GTS on U50-induced analgesia was completely blocked by flumazenil and picrotoxin but not bicuculline. Taken together, it is evident that benzodiazepine receptors and GABAA-gated chloride channels are involved in the attenuation of U50-induced analgesia by GTS. Several lines of study indicates that descending GABAergic system play an important role in modulation of mu- and kappa opioid-induced analgesia [8–11]. However, the role of benzodiazepine–GABAAergic mechanisms in the development of tolerance to U50-induced analgesia is not known. In this study, we found that diazepam inhibits tolerance to U50induced analgesia and this effect is completely blocked by flumazenil, picrotoxin and bicuculline. These findings indicate that diazepam by acting on benzodiazepine–GABAA receptor complex inhibits tolerance to kappa opioid-induced analgesia. These findings not only indicate that the activation of benzodiazepine – GABAergic transmission inhibits tolerance to kappa opioid-induced analgesia, but also suggest that the benzodiazepine– GABAA receptor complex is involved in the inhibitory effect of GTS on tolerance to kappa opioid-induced analgesia. This is evidenced by the findings that flumazenil and picrotoxin blocked the inhibition of tolerance to U50-induced analgesia by GTS. This is in line with the findings that GTS block the uptake of GABA and interferes with the binding of GABAA ligands to rat brain membrane [18,19]. However, bicuculline blocked the attenuation of U50induced analgesia and inhibition of tolerance to its analgesia by diazepam but not GTS. These findings clearly indicate that GTS and diazepam differentially modulate GABAA receptor. It has been reported that diazepam interacts indiscriminately with all benzodiazepine–sensitive GABAA receptor subtypes (a1,a2,a3 and a5) and produces antianxiety, anticonvulsant effects and myorelaxation, with side effects including anterograde amnesia, impairment of motor coordination and potentiation of ethanol effects [23]. There is increasing evidence that agents that can selectively activate GABAA receptors subtype may produce the beneficial effects of diazepam without the unwanted effects [23,24]. Previous studies indicate that ginseng like diazepam produces antianxiety effects in various animal models of anxiety [20]. Eventhough, ginseng has some of the effects of diazepam, it does not cause impairment of motor activity, induce undue sedation or interfere with the memory engram [25,26]. In the present study, it was observed that unlike diazepam, the effects of GTS were insensitive to bicuculline. However, the effect of GTS and diazepam on U50-induced analgesia were
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blocked by flumazenil and picrotoxin. These findings lead us to speculate that GTS may be acting at GABAA subtypes, which are insensitive to bicuculline. Further studies are needed to confirm this speculation. The bicuculline-insensitive actions of GTS may be the reason for lack of some of the side effects of diazepam. Paradoxically, bicuculline per se inhibited the tolerance to U50-induced analgesia. However, when bicuculline is combined with diazepam, the inhibitory effect of diazepam on tolerance to U50-induced analgesia is lost. It has been reported that bicuculline apart from GABAA receptor antagonist effects, produces many other non-GABA mediated effects [27]. Thus, the inhibitory effect of bicuculline on tolerance to U50-induced analgesia may be due its non-GABA receptor-mediated effects. Further studies are needed for investigating the molecular mechanisms of this phenomenon. Recent studies indicates that GTS and some of its saponins attenuate kappa opioid-induced analgesia by acting at spinal and supraspinal sites [2]. Thus, the attenuation effect of systemic treatment of GTS on U50-induced analgesia may, atleast in part, be due to an action of absorbed GTS at the spinal and supraspinal sites. It may be possible that GTS by acting on benzodiazepine receptors and GABAA –gated chloride channels at spinal and supraspinal sites attenuate U50-induced analgesia and inhibits tolerance to its analgesia in mice. Several lines of study indicate that 5-HT play a major role in the U50-induced analgesia and its tolerance to analgesia in mice tail–flick test [22,28]. GTS is reported to attenuate U50-induced analgesia through serotonergic mechanisms [3]. In addition, it is demonstrated recently that activation of descending noradrenergic and serotonergic system partly depends on the activation of GABAergic or/ and glycinergic interneurons [29]. Thus, GTS may attenuate U50-induced analgesia and inhibit tolerance to its analgesia by activation of GABAergic inhibitory interneurons there by decreasing the levels of 5-HT. This is supported by the fact that depletion of serotonin with p–chlorophenylalanine (p–CPA) attenuates U50-induced analgesia and inhibits tolerance to its analgesia in mice (unpublished data). Involvement of other mechanisms in attenuation of U50-induced analgesia and inhibition of tolerance to U50-induced analgesia by GTS and diazepam cannot be ruled out. It might be possible that GTS and diazepam by acting on opioid receptors may attenuate U50induced analgesia and inhibit tolerance to U50-induced analgesia. Recent studies indicate that diazepam does not show any affinity to mu- and kappa-opioid receptors in mouse brain preparation [30]. However, to date no reports is available on the effect of GTS on binding of opioid ligands to opioid receptors. Hence, to find whether GTS has any affinity to opioid receptors, the affect of GTS on binding of [3H]naloxone to mouse brain membrane was studied. The findings indicated that GTS has no affinity to opioid receptors suggesting that GTS may affect opioid-induced effects by indirect action. It might be possible that diazepam modulate the opioid-induced analgesia and its tolerance to analgesia by modulating opioid receptors [17,31] and endogenous opioid peptides [14,32] on chronic treatment. However, to date there is no information available on the effect of GTS on opioid receptors and opioid peptides. The other possible mechanisms may be that GTS and diazepam may affect similar transduction mechanisms, which are involved in the mediating the effects of opioids. Ginseng and diazepam were demonstrated to inhibit the cAMP in different test systems [33–35].
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Conclusions In summary, the present findings indicate that GTS and diazepam on acute treatment attenuate U50-induced analgesia. Further, the study for the first time demonstrated that GTS attenuates U50-induced analgesia by acting on benzodiazepine receptors and GABAA –gated chloride channels. In addition, it was demonstrated that diazepam on chronic treatment inhibits tolerance to U50-induced analgesia. Further, the study demonstrated that diazepam by acting on benzodiazepine–GABAA receptor complex inhibit tolerance to U50-induced analgesia suggesting the crucial role of that benzodiazepine–GABAA receptor complex in the development of tolerance to U50-induced analgesia. Moreover, the results of the study for the first time demonstrated that GTS by acting on benzodiazepine receptors and GABAA – gated chloride channels inhibit tolerance to U50-induced analgesia. Acknowledgments Council of Scientific and Industrial Research, New Delhi, INDIA is gratefully acknowledged for grant of senior research fellowship to K V. S. N. The authors are grateful to M/s. Pharmacia and Upjohn, USA. and Korea Ginseng and Tobacco Research Institute, Taejon, Korea for the supply of U-50,488H and ginseng total saponin, respectively as gift samples. The gift samples of diazepam and flumazenil from Ranbaxy Laboratories Limited (India), F.Hoffman -La Roche AG (CH-4070, Basel), respectively are highly acknowledged. References 1. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochemical Pharmacology 1999;58:1685 – 93. 2. Suh HW, Song DK, Huh SO, Kim YH. Modulatory role of ginsenosides injected intrathecally or intracerebroventrically in the production of antinociception induced by kappa-opioid receptor agonist administered intracerebroventrically in the mouse. Planta Medica 2000;66:412 – 7. 3. Kim HS, Oh KW, Rheu HM, Kim SH. Attenuation of U-50,488H-induced antinociception by ginseng total saponins is dependent on serotonergic mechanisms. Pharmacology Biochemistry and Behavior 1992;42: 587 – 93. 4. Choi S, Jung SY, Rhim H, Jeong SW, Lee SM, Nah SY. Evidence that ginsenosides prevent the development of opioid tolerance at the central nervous system. Life Sciences 2000;67:969 – 75. 5. Kim HS, Oh KW, Park WK, Yahamano S, Toki S. Effects of Panax ginseng on the development of morphine tolerance and dependence. Korean Journal of Ginseng Science 1987;11:182 – 90. 6. Bhargava HN, Ramarao P. The effect of Panax ginseng on the development of tolerance to the pharmacological actions of morphine in the rat. General Pharmacology 1991;22:429 – 34. 7. Nemmani KVS, Ramarao P. Effect of ginseng saponins on U-50,488H analgesia and its tolerance to analgesia in mice. Pharmacy and Pharmacology Communications 2000;6:527 – 32. 8. Fennesy MR, Sawynok J. The effect of benzodiazepines on the analgesic activity of morphine and sodium salicylate. Archives Internationales De Pharmacodynamic Et De Therapie 1973;204:77 – 8. 9. Mantegazza P, Parenti M, Tammiso R, Vita P, Zambotti F, Zonta N. Modification of the antinociceptive effect of morphine by centrally administered diazepam and midazolam. British Journal of Pharmacology 1982;75: 569 – 72.
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10. Palaoglu O, Ayhan IH. The possible role of benzodiazepine receptors in morphine analgesia. Pharmacology Biochemistry and Behavior 1986;25:215 – 7. 11. Huong NTT, Matsumoto K, Yamasaki K, Duc NM, Nham NT, Watanabe H. Majonoside – R2, a major constituent of Vietnamese ginseng, attenuates opioid-induced antinociception. Pharmacology Biochemistry and Behavior 1997;57:285 – 91. 12. Haefely W, Polc P. Physiology of GABA enhancement of benzodiazepines and barbiturates. In: Olesn RW, Venter JC, editors. benzodiazepine – GABA receptors and chloride ion channels; structure and functional properties. New York: Alan R. Liss, 1986. pp. 97 – 133. 13. Huong NTT, Matsumoto K, Yamasaki K, Duc NM, Nham NT, Watanabe H. The possible involvement of GABAA systems in the antinarcotic effect of majonoside – R2, a major constituent of vietnamese ginseng, in mice. Japanese Journal of Pharmacology 1996;71:345 – 9. 14. Sribanditmongkol P, Sheu MJ, Tejwani GA. Inhibition of morphine tolerance and dependence by diazepam and its relation to the CNS Met-enkephalin levels. Brain Research 1994;645:1 – 12. 15. Tejwani GA, Rattan AK, Sribanditmongkol P, Sheu MJ, Zuniga J, McDonald JS. Inhibition of morphineinduced tolerance and dependence by a benzodiazepine receptor agonist midazolam in the rat. Anesthesia Analogy 1993;76:1052 – 60. 16. Sheu MJ, Sribanditmongkol P, Santosa DN, Tejwani GA. Inhibition of morphine tolerance and dependence by diazepam and its relation to cyclic AMP levels in discrete rat brain regions and spinal cord. Brain Research 1995;775:31 – 7. 17. Tejwani GA, Sheu MJ, Sribanditmongkol P, Satyapriya A. Inhibition of morphine tolerance and dependence by diazepam and its relation to m-opioid receptors in the rat brain and spinal cord. Brain Research 1998;797: 305 – 12. 18. Tsang D, Yeung HW, Tso WW, Peck H. Ginseng saponins: Influence of neurotransmitter uptake in rat brain synaptosomes. Planta Medica 1985;51:221 – 4. 19. Kimara T, Saunders PA, Kim HS, Rheu HM, Oh KW, Ho IK. Interactions of ginsenosides with ligandbindings of GABA (A) and GABA (B) receptors. General Pharmacology 1994;25:193 – 9. 20. Bhattacharya SK, Mitra SK. Anxiolytic activity of Panax ginseng roots: an experimental study. Journal of Ethnopharmacology 1991;34:87 – 92. 21. Yoburn BC, Billings B, Duttaroy A. Opioid receptor regulation in mice. Journal of Pharmacology and Experimental Therapeutics 1993;265:314 – 20. 22. Von Voigtlander PF, Lewis RA, Neff GL. Kappa opioid analgesia is dependent on serotonergic mechanisms. Journal of Pharmacology and Experimental Therapeutics 1984;231:270 – 4. 23. Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H. Benzodiazepine actions mediated by specific g – aminobutyric acidA receptor subtypes. Nature 1999;401: 796 – 800. 24. Mehta AK, Ticku MJ. An update on GABAA receptors. Brain Research Reviews 1999;29:196 – 217. 25. Owen RT. Ginseng: A pharmacological profile. Drugs of Today 1981;7:331 – 6. 26. Bhattacharya SK, Tandon R, Mitra SK, Bajpai HS. Panax ginseng: A pharmacological and clinical appraisal. Journal of Internal Medicine 1990;2:17 – 21. 27. Seutin V, Johnson SW. Recent advances in the pharmacology of quaternary salts of bicuculline. Trends in Pharmacological Sciences 1999;20:268 – 70. 28. Ho BY, Takemori AE. Serotonergic involvement in the antinociceptive action of and the development of tolerance to the kappa-opioid receptor agonist, U-50, 488H. Journal of Pharmacology and Experimental Therapeutics 1989;250:508 – 14. 29. Peng YB, Lin Q, Willis WD. Effects of GABA and glycine receptor antagonists on electrical activity and PAG-induced inhibition of rat dorsal horn neurons. Brain Research 1996;736:189 – 201. 30. Rosland JH, Hole K. Benzodiazepine-induced attenuation of opioid antinociception may be abolished by spinalization or blockade of the benzodiazepine receptor. Pharmacology Biochemistry and Behavior 1990;37: 505 – 9. 31. Beliaev NA, Petrichenko OB. Effect of diazepam on the specific binding of 3H-(D-Ala2, D-Leu5)- enkephalin by striatal membranes in the rat. Pharmacology and Toxicology 1988;51:18 – 21.
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Role of benzodiazepine–GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice Kumar V.S. Nemmani, P. Ramarao* Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Phase - X, S.A.S. Nagar - 160 062 (Pb), India Received 27 June 2001; accepted 1 October 2001
Abstract In the present study, the role of benzodiazepine – GABAA receptor complex in the attenuation of U50,488H (U50), a selective kappa opioid agonist-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin (GTS) was investigated using the mice tail –flick test. The intraperitoneal (i.p.) treatment of GTS (100 and 200 mg/kg) and diazepam (0.1 – 1 mg/kg) dosedependently attenuated the U50 (40 mg/kg, i.p.)-induced analgesia. GTS (0.001 – 10 mg/ml) did not alter binding of [3H]naloxone to mice whole brain membrane. The attenuation effect of GTS (100 mg/ kg) and diazepam (0.5 mg/kg) on U50-induced analgesia was blocked by flumazenil (0.1 mg/kg, i.p.), a benzodiazepine receptor antagonist, and picrotoxin (1 mg/kg, i.p.), a GABAA-gated chloride channel blocker. However, bicuculline (1 mg/kg, i.p.), a GABAA receptor antagonist blocked the attenuation effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on U50-induced analgesia. Chronic treatment (day 4 – day 6) of GTS (50 –200 mg/kg) and diazepam (0.1 – 1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) on chronic treatment blocked the inhibitory effect of GTS (100 mg/kg) and diazepam (0.5 mg/kg) on tolerance to U50-induced analgesia. On the other hand, chronic treatment of bicuculline (1 mg/kg) blocked the inhibitory effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on tolerance to U50induced analgesia. In conclusion, the findings suggest that GTS attenuates U50-induced analgesia and
* Corresponding author. Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Phase - X, S.A.S. Nagar - 160 062 (Pb), India. Tel.: +91-172-214-683x2043; fax: +91-172-214-692. E-mail address: [email protected] (P. Ramarao). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 4 9 6 - 0
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inhibits tolerance to its analgesia and this action involves benzodiazepine receptors and GABAA-gated chloride channels. D 2002 Elsevier Science Inc. All rights reserved. Keywords: ginseng total saponin; diazepam; kappa opioid agonist; analgesia; tolerance; benzodiazepine – GABAA receptor complex
Introduction The use of Panax ginseng, C. A. Meyer (Aralaciaceae) root as a folklore medicine in the far eastern countries goes back to thousands of years. Many studies indicate that ginseng saponins are the major active constituents of Panax ginseng [1]. Ginseng total saponin (GTS), a mixture of total saponins of Panax ginseng and some of its saponins have been demonstrated to attenuate morphine, a mu opioid- and U-50,488H (U50), a selective kappa opioid-induced analgesia in the mice tail–flick test [2,3]. Further, GTS and ginseng extracts have been reported to inhibit tolerance to morphine- induced analgesia in rodents [4–6]. Recently, we reported that GTS, panaxadiol and panaxatriol inhibit tolerance to U50-induced analgesia in mice [7]. However, the mechanism(s) by which GTS is able to attenuate opioid-induced analgesia and inhibit tolerance to its analgesia are not known. Several studies indicate that GABAergic mechanisms play a role in the modulation of muand kappa opioid-induced analgesia [8–11]. Diazepam, a benzodiazepine receptor agonist is well known to produce many of its effects through enhancement of GABAergic neurotransmission [12]. Further, the attenuation effect of diazepam on mu-and kappa opioidinduced analgesia was blocked by flumazenil, a benzodiazepine receptor antagonist and picrotoxin, a GABAA receptor-gated chloride channel blocker suggesting the involvement of benzodiazepine – GABAAergic mechanisms in attenuation of opioid-induced analgesia [11,13]. In addition, recent studies indicated that diazepam and midazolam inhibit tolerance to morphine-induced analgesia [14–17]. Ginseng extracts block the uptake of GABA into rat brain synaptosomes [18]. Moreover, GTS and some of its saponins are reported to alter the binding of GABAA [19]. In addition, ginseng is demonstrated to have diazepam like activity [20]. Since GTS has diazepam like activity, block the uptake of GABA and alter the binding of GABAA ligands, we hypothesise that GTS possibly attenuates U50-induced analgesia and inhibits tolerance to its analgesia via benzodizepine–GABAAergic mechanisms. Hence, in the present studies the possible role of benzodiazepine –GABAAergic mechanisms in attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS are investigated using the selective antagonists of benzodiazepine–GABAA receptor complex in the mice tail–flick test. Methods Animals Swiss male mice (Central Animal Facility, NIPER, India) weighing 20–26 g were housed five to a cage in a room with controlled temperature (22 ± 2 C), humidity (50 ± 10%) and
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light (06.00–18.00 h). Food and water were made available ad libitum. All experiments were performed between 10.00 and 16.00 h to minimize diurnal variations. Measurement of analgesic response by tail–flick test in mice The analgesic response was determined as described previously by modified tail–flick test [7]. Briefly, the tail flick latencies to thermal stimulation (radiant heat) were determined at 0, 30, 60, 90, 120 and 180 min after administration of U50. The basal latencies were found to 5–7 sec. A cutoff time of 20 sec was followed to prevent any injury to the tail. The percent maximum possible effect (%MPE) was calculated using the formula % MPE ¼ ðPost latency Basal latencyÞ=ðCutoff latency Basal latencyÞ 100
ð1Þ
A graph is plotted as Mean % MPE ± S.E.M. vs time. The area under curve (AUC 0 – 180 min.) of %Mean % MPE ± S.E.M. vs time plot was calculated for different groups and the data was subjected to statistical analysis. Induction and assessment of tolerance to U50-induced analgesia in mice Mice were rendered tolerant to U50 by administration of U50 (40 mg/kg) daily in the morning and evening for six days [7]. Mice serving as control were administered similarly with vehicle. On day 7 analgesic response was assessed as described above in separate groups of mice challenging with U50 (40 mg/kg). The AUC 0 – 180 min was calculated for each mouse and subjected to statistical analysis. Determination of displacement of [3H]naloxone (4nM) binding to mice whole brain by GTS Binding studies were performed with slight modification of the method described by Yoburn et al [21]. Mice were sacrificed and whole brains were removed, weighed and homogenised in 20 volumes of ice-cold 50 mM Tris buffer (PH 7.6). The homogenates were then centrifuged (49000 g, 4 C, and 15 min) and the pellet was resuspended in buffer and incubated (30 min, 25 C). Homogenate was again centrifuged and supernatant was removed and suspended in 20 volumes of Tris buffer. An aliquot of brain homogenate containing 250– 300 mg of protein was assayed in triplicate containing 4 nM of [3H]naloxone. The amount of ligand displaced by GTS was determined at the concentration range of 0.001–10 mg/ml. Similarly, a parallel study was conducted in triplicate with naloxone (0.001 nM–1 mM). Homogenate was incubated for 90 min at 25 C. To the homogenate ice-cold buffer was added and then filtered using Brandel filtration unit with GF/B whatman filter paper. The tubes were washed with buffer, filter papers were then transferred to scintillation vials and cocktail was added. After overnight saturation, the samples were counted using Wallac 1407 b-counter (Efficiency 49%).
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Drugs The substances used in the study were U50 (Pharmacia and Upjohn Company, Kalamazoo, USA), GTS (Korea Ginseng and Tobacco Research Institute, Taejon, Korea) which is mixture of ginsenosides and the ratios of various ginsenosides were Rb1 (18.26%), Rb2 (9.07%), Rc (9.65%), Rd (8.24%), Re (9.28%), Rf (3.48%), Rg1 (6.42%), Rg2 (3.63%), Rg1 (4.70%), R0 (3.82%), Ra (2.91%) and other minor ginsenosides, diazepam (Ranbaxy Laboratories, Dewas, India), flumazenil (F.Hoffman -La Roche AG, Basel, Switzerland), [N-allyl2,3-3H]naloxone (Amersham Life science, England), naloxone (Sigma Chemical, St. Louis, USA), picrotoxin (Sigma Chemical, St. Louis, USA) and bicuculline (Sigma Chemical, St. Louis, USA). U50 and GTS were dissolved in distilled water. Diazepam and flumazenil were dissolved in 2% DMSO-water. Picrotoxin was dissolved in 2% ethanol–water whereas, bicuculline was initially dissolved in few drops of 1N HCl and diluted with distilled water. All substances were prepared freshly and administered i.p. in a volume of 10 ml/kg of body weight. Treatment schedule In the acute study, GTS and diazepam were administered 4 hr and 30 min prior to U50 administration, respectively. Whereas, flumazenil, picrotoxin and bicuculline were administered 1 hr prior to U50 administration. In tolerance studies, GTS and diazepam were administered chronically (day 4–day 6) once daily 4 hr and 30 min prior to evening dose of U50, respectively. Similarly, flumazenil, picrotoxin and bicuculline were administered chronically (day 4–day 6) 1 hr prior to evening dose of U50. Statistical analysis The data was analyzed by one-way analysis of variance (one-way ANOVA) followed by a post-hoc multiple comparison test (Scheffe’s S–test). A value of p< 0.05 was considered to be significant.
Results Effect of GTS and diazepam on U50-induced analgesia in mice The effect of GTS (50, 100 and 200 mg/kg) and diazepam (0.1, 0.5 and 1 mg/kg) on timecourse of action and AUC 0 – 180 min of U50-induced analgesia in mice tail–flick test is shown in Fig. 1. U50 (40 mg/kg, i.p.) produced analgesia with peak maximal effect at 30 min after the administration. GTS (50–200 mg/kg) and diazepam (0.1–1 mg/kg) per se did not produce analgesia (data not shown). GTS (100 and 200 mg/kg) attenuated the peak maximal response of U50-induced analgesia (Fig. 1A). The % decrease in analgesic response to U50 (expressed as AUC 0 – 180 min) by GTS (100 and 200 mg/kg) were 45.5 ± 3.6 and 58.6 ± 3.9,
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Fig. 1. Effect of GTS (50, 100 and 200 mg/kg, i.p.) and diazepam (0.1, 0.5 and 1 mg/kg, i.p.) on time-course of action and AUC 0 – 180min of U50 (40 mg/kg, i.p.)-induced analgesia in the mice tail – flick test. GTS (A) and diazepam (B) were treated at 4 hr and 30 min prior to U50 administration, respectively. # P< 0.05 vs Vehicle.
respectively (Fig. 1A). Diazepam (0.1, 0.5 and 1 mg/kg) also dose-dependently attenuated the U50-induced analgesia (Fig. 1B). The % decrease in analgesic response to U50-induced analgesia by diazepam (0.1, 0.5 and 1 mg/kg) were 43.0 ± 3.2, 55.3 ± 6.2 and 80.2 ± 3.9, respectively (Fig. 1B). Effect of flumazenil, picrotoxin and bicuculline on the attenuation of U50-induced analgesia by GTS and diazepam The effect of flumazenil, picrotoxin and bicuculline on attenuation of U50-induced analgesia by GTS and diazepam is shown in Fig. 2. GTS (100 mg/kg) and diazepam
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Fig. 2. Effect of flumazenil (0.1 mg/kg, i.p.), picrotoxin (1 mg/kg, i.p.), and bicuculline (1 mg/kg, i.p.) on attenuation of U50 (40 mg/kg)-induced analgesia by GTS (100 mg/kg, i.p., 4 hr prior) and diazepam (0.5 mg/kg, i.p., 30 min prior) in mice. Flumazenil (A), picrotoxin (B) and bicuculline (C) were treated one hr prior to U50 administration. Unfilled bar indicates control mice; Hatched bars indicate treated mice; All the values are expressed as Mean ± S. E. M., n = 5. * P< 0.05 vs control; # P< 0.05 vs respective GTS or diazepam.
(0.5 mg/kg) attenuated the U50 (40 mg/kg)-induced analgesia. Flumazenil (0.1 mg/kg), picrotoxin (1 mg/kg) and bicuculline (1 mg/kg) per se did not produce analgesia (data not shown) or altered U50-induced analgesia (Fig. 2). However, flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) blocked the attenuation of U50-induced analgesia by GTS and
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Fig. 3. Effect of GTS (0.001 – 10 mg/ml) and naloxone (0.001 nM – 1mM) on binding of 4 nM [3H]naloxone to mice whole brain membrane. All the values are expressed as Mean ± S. E. M., n = 3.
diazepam (Fig 2 A and B). On the other hand, bicuculline (1 mg/kg) blocked the attenuation of U50-induced analgesia by diazepam but not GTS (Fig. 2 C). Table 1 Effect of GTS and diazepam on tolerance to U50-induced analgesia in mice Treatment Group
Analgesic Response to U50 AUC Mean ± S.E.M (n = 5)
GTS Treatment groups Vehicle + Vehicle U50 Treated + Vehicle U50 Treated + GTS (50) U50 Treated + GTS (100) U50 Treated + GTS (200)
2731.2 591.9 1542.5 2662.0 2763.9
± ± ± ± ±
103.6 128.0 * 120.1 # 112.9 # 119.6 #
Diazepam treatment groups Vehicle + Vehicle U50 Treated + Vehicle U50 Treated + Diazepam (0.1) U50 Treated + Diazepam (0.5) U50 Treated + Diazepam (1)
2920.8 501.9 1791.7 2625.4 2721.8
± ± ± ± ±
135.8 94.9 * 200.4# 263.2# 175.2#
0 – 180 min
@ Mice were treated with U50 (40 mg/kg, i.p.) twice daily for six days. GTS (50, 100 and 200 mg/kg, 4 h prior) and diazepam (0.1, 0.5 and 1 mg/kg, 30 min prior) were administered once daily on day 4 - day 6 prior to the evening dose of U50. Analgesic response to U50 (40 mg/kg) was determined on day 7 in mice from all groups. * p < 0.05 vs respective Vehicle + Vehicle group; # p < 0.05 vs respective U50 Treated + Vehicle group.
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Fig. 4. Effect of flumazenil (0.1 mg/kg, i.p.), picrotoxin (1 mg/kg, i.p.), and bicuculline (1 mg/kg, i.p.) on inhibition of tolerance to U50 (40 mg/kg)-induced analgesia by GTS (100 mg/kg, i.p.) and diazepam (0.5 mg/kg, i.p.) in mice. Mice were rendered tolerant to U50 by twice daily administration of U50 (40 mg/kg, i.p.) for six days. GTS and diazepam were treated once daily on day 4 - day 6 at 4hr and 30 min prior to evening dose of U50, respectively. Whereas, flumazenil (A), picrotoxin (B) and bicuculline (C) were treated one hr prior to evening dose of U50 on day 4 - day 6. The analgesic response to U50 (40 mg/kg, i.p.) was determined on day 7. Unfilled bar indicates U50naive and hatched bars indicate U50-tolerant. All the values are expressed as Mean ± S. E. M., n = 5. * P< 0.05 vs U50-naive; # P< 0.05 vs U50-tolerant; @ P< 0.05 vs U50-tolerant + respective GTS or diazepam.
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Effect of GTS on [3H]naloxone binding to mice whole brain The effect of GTS on binding of [3H]naloxone to mice whole brain membrane is shown in Fig. 3. As shown in Fig. 3, GTS did not alter the [3H]naloxone binding. On the other hand, naloxone displaced the [3H]naloxone binding with IC50 of 0.52 nM ± 0.07. Effect of chronic treatment of GTS and diazepam on tolerance to U50-induced analgesia Twice daily administration of U50 (40 mg/kg) for six days resulted in the development of tolerance to U50-induced analgesia. As shown in the Table 1, chronic administration of U50 decreased the analgesic response to U50 by about five folds when compared to that of vehicle administered group. Chronic treatment of GTS (50, 100 and 200 mg/kg) and diazepam (0.1, 0.5 and 1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia (Table 1). Chronic treatment of GTS (50, 100 and 200 mg/kg) to U50-tolerant mice restored the analgesic response to U50 to 56.5 % ± 4.4, 97.5 % ± 4.1 and 101.2 % ± 4.4, respectively. Moreover, chronic administration of diazepam (0.1, 0.5 and 1 mg/kg) to U50-tolerant mice also restored the analgesic response to U50 to 58.4 % ± 8.0, 89.9 % ± 9.0 and 93.2 % ± 6.0, respectively in mice (Table 1). Effect of flumazenil, picrotoxin and bicuculline on the inhibition of tolerance to U50-induced analgesia by GTS and diazepam The effect of flumazenil, picrotoxin and bicuculline on the inhibition of tolerance to U50induced analgesia by GTS and diazepam is shown in Fig. 4. Twice daily administration of U50 (40 mg/kg) for six days resulted in the development of tolerance to U50-induced analgesia (Fig. 4). As shown in the Fig. 4, chronic treatment of GTS (100 mg/kg), and diazepam (0.5 mg/kg) inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/ kg) and picrotoxin (1 mg/kg) on chronic treatment did not alter the tolerance to U50-induced analgesia. However, chronic treatment of flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) blocked the inhibition of tolerance to U50-induced analgesia by GTS and diazepam (Fig. 4 A and B). On other hand, bicuculline per se inhibited the tolerance to U50-induced analgesia. In addition, bicuculline blocked the inhibition of tolerance to U50-induced analgesia by diazepam but not GTS (Fig. 4 C).
Discussion U50, a selective kappa opioid agonist appears to produce analgesia by activation of descending pain inhibitory system in mice tail–flick test [22]. Recent studies indicate that intracerebroventricularly (i.c.v.) and intrathecaly (i.t.) administered GTS and its saponins dose-dependently attenuate kappa opioid-induced analgesia in the mice tail–flick test [2]. Further, i.c.v. administered GTS inhibits tolerance to morphine-induced analgesia [4]. We reported recently that systemic treatment of GTS and its two major fractions viz panaxadiol
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and panaxatriol inhibit tolerance to U50-induced analgesia [7]. However, the mechanism(s) by which GTS exerts its effects on opioid-induced analgesia and its tolerance to analgesia are not known. In an attempt to determine the mechanism(s) of attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS, the effect of antagonists of benzodiazepine– GABAA receptor complex on attenuation of U50-induced analgesia and inhibition of tolerance to its analgesia by GTS were investigated in the present study. In consistent with the literature, we found that systemic treatment of GTS dose-dependently attenuates U50induced analgesia [3]. In this study, we also found that systemic treatment of diazepam dosedependently attenuates U50-induced analgesia, and that the effect of diazepam was blocked by flumazenil, picrotoxin and bicuculline. Thus these findings support the idea that enhancement of benzodiazepine–GABAergic transmission negatively modulates the kappa opioid induced analgesia [11,13]. In this study, we also found that the attenuation effect of GTS on U50-induced analgesia was completely blocked by flumazenil and picrotoxin but not bicuculline. Taken together, it is evident that benzodiazepine receptors and GABAA-gated chloride channels are involved in the attenuation of U50-induced analgesia by GTS. Several lines of study indicates that descending GABAergic system play an important role in modulation of mu- and kappa opioid-induced analgesia [8–11]. However, the role of benzodiazepine–GABAAergic mechanisms in the development of tolerance to U50-induced analgesia is not known. In this study, we found that diazepam inhibits tolerance to U50induced analgesia and this effect is completely blocked by flumazenil, picrotoxin and bicuculline. These findings indicate that diazepam by acting on benzodiazepine–GABAA receptor complex inhibits tolerance to kappa opioid-induced analgesia. These findings not only indicate that the activation of benzodiazepine – GABAergic transmission inhibits tolerance to kappa opioid-induced analgesia, but also suggest that the benzodiazepine– GABAA receptor complex is involved in the inhibitory effect of GTS on tolerance to kappa opioid-induced analgesia. This is evidenced by the findings that flumazenil and picrotoxin blocked the inhibition of tolerance to U50-induced analgesia by GTS. This is in line with the findings that GTS block the uptake of GABA and interferes with the binding of GABAA ligands to rat brain membrane [18,19]. However, bicuculline blocked the attenuation of U50induced analgesia and inhibition of tolerance to its analgesia by diazepam but not GTS. These findings clearly indicate that GTS and diazepam differentially modulate GABAA receptor. It has been reported that diazepam interacts indiscriminately with all benzodiazepine–sensitive GABAA receptor subtypes (a1,a2,a3 and a5) and produces antianxiety, anticonvulsant effects and myorelaxation, with side effects including anterograde amnesia, impairment of motor coordination and potentiation of ethanol effects [23]. There is increasing evidence that agents that can selectively activate GABAA receptors subtype may produce the beneficial effects of diazepam without the unwanted effects [23,24]. Previous studies indicate that ginseng like diazepam produces antianxiety effects in various animal models of anxiety [20]. Eventhough, ginseng has some of the effects of diazepam, it does not cause impairment of motor activity, induce undue sedation or interfere with the memory engram [25,26]. In the present study, it was observed that unlike diazepam, the effects of GTS were insensitive to bicuculline. However, the effect of GTS and diazepam on U50-induced analgesia were
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blocked by flumazenil and picrotoxin. These findings lead us to speculate that GTS may be acting at GABAA subtypes, which are insensitive to bicuculline. Further studies are needed to confirm this speculation. The bicuculline-insensitive actions of GTS may be the reason for lack of some of the side effects of diazepam. Paradoxically, bicuculline per se inhibited the tolerance to U50-induced analgesia. However, when bicuculline is combined with diazepam, the inhibitory effect of diazepam on tolerance to U50-induced analgesia is lost. It has been reported that bicuculline apart from GABAA receptor antagonist effects, produces many other non-GABA mediated effects [27]. Thus, the inhibitory effect of bicuculline on tolerance to U50-induced analgesia may be due its non-GABA receptor-mediated effects. Further studies are needed for investigating the molecular mechanisms of this phenomenon. Recent studies indicates that GTS and some of its saponins attenuate kappa opioid-induced analgesia by acting at spinal and supraspinal sites [2]. Thus, the attenuation effect of systemic treatment of GTS on U50-induced analgesia may, atleast in part, be due to an action of absorbed GTS at the spinal and supraspinal sites. It may be possible that GTS by acting on benzodiazepine receptors and GABAA –gated chloride channels at spinal and supraspinal sites attenuate U50-induced analgesia and inhibits tolerance to its analgesia in mice. Several lines of study indicate that 5-HT play a major role in the U50-induced analgesia and its tolerance to analgesia in mice tail–flick test [22,28]. GTS is reported to attenuate U50-induced analgesia through serotonergic mechanisms [3]. In addition, it is demonstrated recently that activation of descending noradrenergic and serotonergic system partly depends on the activation of GABAergic or/ and glycinergic interneurons [29]. Thus, GTS may attenuate U50-induced analgesia and inhibit tolerance to its analgesia by activation of GABAergic inhibitory interneurons there by decreasing the levels of 5-HT. This is supported by the fact that depletion of serotonin with p–chlorophenylalanine (p–CPA) attenuates U50-induced analgesia and inhibits tolerance to its analgesia in mice (unpublished data). Involvement of other mechanisms in attenuation of U50-induced analgesia and inhibition of tolerance to U50-induced analgesia by GTS and diazepam cannot be ruled out. It might be possible that GTS and diazepam by acting on opioid receptors may attenuate U50induced analgesia and inhibit tolerance to U50-induced analgesia. Recent studies indicate that diazepam does not show any affinity to mu- and kappa-opioid receptors in mouse brain preparation [30]. However, to date no reports is available on the effect of GTS on binding of opioid ligands to opioid receptors. Hence, to find whether GTS has any affinity to opioid receptors, the affect of GTS on binding of [3H]naloxone to mouse brain membrane was studied. The findings indicated that GTS has no affinity to opioid receptors suggesting that GTS may affect opioid-induced effects by indirect action. It might be possible that diazepam modulate the opioid-induced analgesia and its tolerance to analgesia by modulating opioid receptors [17,31] and endogenous opioid peptides [14,32] on chronic treatment. However, to date there is no information available on the effect of GTS on opioid receptors and opioid peptides. The other possible mechanisms may be that GTS and diazepam may affect similar transduction mechanisms, which are involved in the mediating the effects of opioids. Ginseng and diazepam were demonstrated to inhibit the cAMP in different test systems [33–35].
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Conclusions In summary, the present findings indicate that GTS and diazepam on acute treatment attenuate U50-induced analgesia. Further, the study for the first time demonstrated that GTS attenuates U50-induced analgesia by acting on benzodiazepine receptors and GABAA –gated chloride channels. In addition, it was demonstrated that diazepam on chronic treatment inhibits tolerance to U50-induced analgesia. Further, the study demonstrated that diazepam by acting on benzodiazepine–GABAA receptor complex inhibit tolerance to U50-induced analgesia suggesting the crucial role of that benzodiazepine–GABAA receptor complex in the development of tolerance to U50-induced analgesia. Moreover, the results of the study for the first time demonstrated that GTS by acting on benzodiazepine receptors and GABAA – gated chloride channels inhibit tolerance to U50-induced analgesia. Acknowledgments Council of Scientific and Industrial Research, New Delhi, INDIA is gratefully acknowledged for grant of senior research fellowship to K V. S. N. The authors are grateful to M/s. Pharmacia and Upjohn, USA. and Korea Ginseng and Tobacco Research Institute, Taejon, Korea for the supply of U-50,488H and ginseng total saponin, respectively as gift samples. The gift samples of diazepam and flumazenil from Ranbaxy Laboratories Limited (India), F.Hoffman -La Roche AG (CH-4070, Basel), respectively are highly acknowledged. References 1. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochemical Pharmacology 1999;58:1685 – 93. 2. Suh HW, Song DK, Huh SO, Kim YH. Modulatory role of ginsenosides injected intrathecally or intracerebroventrically in the production of antinociception induced by kappa-opioid receptor agonist administered intracerebroventrically in the mouse. Planta Medica 2000;66:412 – 7. 3. Kim HS, Oh KW, Rheu HM, Kim SH. Attenuation of U-50,488H-induced antinociception by ginseng total saponins is dependent on serotonergic mechanisms. Pharmacology Biochemistry and Behavior 1992;42: 587 – 93. 4. Choi S, Jung SY, Rhim H, Jeong SW, Lee SM, Nah SY. Evidence that ginsenosides prevent the development of opioid tolerance at the central nervous system. Life Sciences 2000;67:969 – 75. 5. Kim HS, Oh KW, Park WK, Yahamano S, Toki S. Effects of Panax ginseng on the development of morphine tolerance and dependence. Korean Journal of Ginseng Science 1987;11:182 – 90. 6. Bhargava HN, Ramarao P. The effect of Panax ginseng on the development of tolerance to the pharmacological actions of morphine in the rat. General Pharmacology 1991;22:429 – 34. 7. Nemmani KVS, Ramarao P. Effect of ginseng saponins on U-50,488H analgesia and its tolerance to analgesia in mice. Pharmacy and Pharmacology Communications 2000;6:527 – 32. 8. Fennesy MR, Sawynok J. The effect of benzodiazepines on the analgesic activity of morphine and sodium salicylate. Archives Internationales De Pharmacodynamic Et De Therapie 1973;204:77 – 8. 9. Mantegazza P, Parenti M, Tammiso R, Vita P, Zambotti F, Zonta N. Modification of the antinociceptive effect of morphine by centrally administered diazepam and midazolam. British Journal of Pharmacology 1982;75: 569 – 72.
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