Life Scieaca, Vol 61, No 4, pp 427d6,195’7 cbpyIi&t~ 1997Elrevier-fnc Rinted in theUSA.All fight8asmcd arz4-32Qsp $17.00 t al zyxwvut PI1 SOO24-3205(97)00400-1 INVOLVEMENT OF SUPBASPINAL GABA RECEPTORS IN MAJONOSIDER2 SUPPRESSION OF CLONIDINE-INDUCED ANTINOCICEPTION IN MICE Nguyen Thi Thu Huongl, Kinzo Matsumotol, Kazuo Yamasaki2 and Hiroshi Watanabe*1 1) Department of Pharmacology, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan 2) Department of Biological Active Substances, Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, l-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan (Rece.ivedin fuml form April 30,19!7) Summary Majonoside-R2 (MR2) is a major constituent of Vietnamese ginseng (Panar viemamensis, Ha et Grushv Araliaceae) that is known to exhibit antagonistic activity against opioid-induced antinociception In this study, we investigated the effect of MR2 on the antinociception caused in mice by the aZadrenoceptor agonist clonidine, and elucidated the role of supraspinal GABAergic systems in this effect of MR2 The systemic administration of clonidine (0.5-2.5 mg/kg, s.c.) dose-dependently suppressed the nociceptive response of mice in the tail-pinch and hot-plate tests The intraperitoneal (i.p.), intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of idazoxan (a selective aZadrenoceptor antagonist) significantly antagonized the antinociceptive effect of clonidine in both tests MR2 administered systemically (1.5-6.2 mg./kg, i.p.) or centrally (5-10 l&mouse, i.c.v or i.t) dose-dependently antagonized the clonidine (1 mg/kg, s.c.)induced antinociception in the tail-pinch test but not in the hot-plate test The effect of i.c.v MR2 on the systemic clonidine-induced antagonistic antinociception in the tail-pinch test was significantly reversed by i.c.v administrations of the selective benzodiazepine receptor antagonist flumazenil (5 pg/mouse) and the GABA, antagonist picrotoxin (0.25 pghnouse) These results suggest that the supraspinal GABA~/benzodiazepine receptors are involved in the antagonistic effect of MR2 on the clonidine-induced antinociception in the tailpinch test in mice Research Corresponding Author: Hiroshi Watanabe, Ph.D., Department of Pharmacology, Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan, FAX: +81-764-34-5056 428 Majonoside-R2 and Antinociception Vol 61, No 4, 1997 Majonoside-R2 (MR2) is one of the major components of Vietnamese ginseng (Panax vietnamensis Ha et Grushv Araliaceae) We have previously demonstrated that psychological stress-induced pathophysiological changes such as antinociception, gastric ulceration and decrease in pentobarbital sleep are prevented by MR2, and that GABAA/benzodiazepine receptor mechanisms are partly involved in this action of MR2 (1, 2) Recent findings from this laboratory indicate that MR2 is also able to antagonize the opioid agonist-induced antinociception in mice tested by tail-pinch test, and that the GABA*/benzodiazepine receptor complex at the supraspinal level mediates this antagonistic effect of MR2 (3) In addition, MR2 was shown to have a suppressive effect on conditioned fear stress-induced antinociception (4) in which the involvement of spinal a2-adrenoceptors has been reported (5) However, it remains unclear whether MR2 can directly modulate a2-adrenoceptor-mediated antinociception by acting at the a2-adrenoceptor sites The descending noradrenergic system is one of the major pain-modulating systems originating from the brainstem; it participates in opioid-induced antinociception in rodents (6-8) This system plays an important role in the transmission of nociceptive information from primary afferent terminals in the spinal cord dorsal horn, and exerts a tonic inhibitory influence on this transmission through a2-adrenoceptors located at pre- and postsynaptic sites of the primary afferent fibers (9-11) Several reports have shown that the aZadrenoceptor agonist clonidine exerts a more potent antinociceptive activity in various nociceptive tests than does morphine (10, 12, 13) Moreover, the activity of the noradrenergic systems involved in the nociceptive response appears to be modulated by GABAergic systems in the brain (14) In the present study, to further clarify the mechanisms underlying the pharmacological activity of MR2, we investigated the effect of MR2 on clonidine-induced antinociception by means of the tail-pinch and hot-plate tests in mice, and elucidated the possible involvement of GABA, receptor systems in the action of MR2 Methods Animals Male ddY mice (Japan SLC, Shizuoka, Japan) were obtained at the age of weeks The mice were housed in groups of 20-25 per cage for at least week before starting the experiments, with free access to food and water Housing conditions were thermostatically maintained at 24 + “C and a relative humidity of 55 * 5% with a 12 h light:dark cycle (lights on: 0730-1930) Each animal was used only once The present studies were conducted in accordance with the standards established by the Guide for the Care and Use of Laboratory Animals of Toyama Medical and Pharmaceutical University Drug adminhintion 1) Systemic administration Clonidine was injected subcutaneously (s.c.) at time MR2 was injected intraperitoneally (i.p.) 30 before clonidine administration The doses, time courses and routes of administration of test drugs were chosen based on the results of previous studies (3) and preliminary experiments All drugs were administered in a constant volume of 0.1 ml/10 g body weight 2) Intracerebroventricular and intrathecal administration of test drugs Intracerebroventricular (i.c.v.) and intrathecal (i.t.) injections of test drugs were performed in a constant total volume of pl, according to the methods of Haley and McCormick (15) and of Hylden and Wilcox (16) respectively When testing antagonism at the supraspinal level, MR2 was co-administered with flumazenil or picrotoxin just before the S.C administration of clonidine M ajonoside-R2and Antimciception Vol 61, No 4, 1997 429 Measurement of the nociceptive response in the tail-pinch and hot-plate tests The nociceptive response in the tail-pinch test was measured according to Haffner’s method as previously reported (1, 7, 17) Briefly, hemostatic forceps (3 mm width, 500 g constant pressure) were applied to the root of the tail, and the latency of the biting response to the forceps was measured The baseline tail-pinch latency was 1.07 f 0.08 s (n=20) No significant differences in the baseline latency between vehicle-treated and test drug-treated groups were observed To prevent tissue damage, a cut-off time of s was selected The nociceptive response in the hot-plate test was measured according to the methods of D’Amour et al (18) and Hunskaar et al (19) Each mouse was placed individually on a metal plate maintained at 55 -t 0.5 “C The latency of nociceptive responses such as hind paw licking, flicking or jumping was measured, and only the mice that showed nociceptive responses within 20 s were used for the experiments The baseline hot-plate latency was 13.47 * 0.93 s (n=17) and no significant differences in the baseline latency between vehicle-treated and test drug-treated groups were observed To prevent tissue damage, a cut-off time of 60 zyxwvutsrqponmlkjihgfedcbaZYXWVUT s was selected zyxwvutsrqponmlkjihgfedcba Drugs MR2 was isolated from the saponin fraction of Vietnamese ginseng as previously described (3, 20) MR2 is water soluble and pH of the MR2 solution is neutral (approximately 7) The following drugs were used: Clonidine HCl, idazoxan HCl and picrotoxin (Sigma Chemical Co., St Louis, MO), flumazenil @rexate@, Yamanouchi Pharmaceutical Co., Tokyo) Test drugs, except for flumazenil, were dissolved in saline Flumazenil was dissolved in saline containing 40% propylene glycol Drug solutions were prepared just before the start of the experiments S&tktical analysis Data are expressed as the mean according to Dewey et al (21) %MPE = (post-drug latency - percent maximum possible pre-drug latency) / (cut-off time - effect (%MPE * SEM) pre-drug latency) x 100 The %MPE was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test or by two-way ANOVA followed by Tukey’s test for multiple comparisons among groups Differences of PcO.05 were considered significant Results Eflects of MR2 on clonidine-induced antinociception The systemic administration of clonidine (0.5-2.5 mgkg, s.c.) significantly prolonged the latency of nociceptive responses in the tail-pinch and hot-plate tests in mice The antinociceptive action of clonidine in the tail-pinch peaked at 15 after administration and then gradually decreased, while the antinociceptive action in the hot-plate test was present throughout the 60-min observation period (Fig 1) Idazoxan (1 mgkg, i.p or 10 pg/mouse, i.c.v or i.t.) significantly antagonized the antinociception caused by clonidine (1 mg/kg, s.c.) in both tests (Figs 1,2 and 3) whereas MR2 (1.5-6.2 mg/kg, i-p or 5-10 y&mouse, i.c.v or i.t.) suppressed the clonidineinduced antinociception in the tail-pinch test but not in the hot-plate test (Figs 1,2 and 3) Supmspinully administered jlumazenil and picrotoxin reversed the suppressing effect of MR2 on the cloruifine-induced antinociception in the tail-pinch test W hen administered i.c.v., neither flumazenil (2.5-5 yg/mouse) nor picrotoxin (0.125-0.25 ug/mouse) had an effect on the antinociceptive action of clonidine (1 mg/kg, s.c.) in the tail-pinch Majonoside-R2 Vol 61, No 4, 1997 and Antisuxiception test However, when i.c.v co-administered with MR2 (10 ug/mouse), significant interactions between MR2 and flumazenil treatment and between MR2 and picrotoxin treatment were observed in the clonidine-induced antinociception [Fw x numazenir(1,39)=8.819, PcO.01; FW x picmtoti(1,40)=5.574, PcO.05, Fig 41 The post hoc test revealed that both i.c.v flumazenil and i.c.v picrotoxin significantly reversed the suppressing effect of MR2 on the clonidine-induced antinociception (B) Hot-plate test (A) Tail-pinch test 100 “‘01 (Al) g 60 I ap 20 4/ , o-‘ + ~ -20 4- -20 30 46 -15 60 100 30 l(W : 45 , 1A: : CLN (1.0) CLN (2.5) 60 T 60 20 -20 15 30 45 60 4, , 15 30 , _ pw 45 60 !$#-.ii;[~ : CLN alone H 0: : MR2 (1 S) MR2 (3.1) A: MR2 (6.2) 15 30 45 60 Time after clonidine 15 30 administration 45 60 (min) Fig The time-courses of systemic clonidine-induced antinociception in the tail-pinch and hot-plate tests in mice, and reversal of clonidine-induced antinociception by systemic idazoxan (IDZ) and majonoside-R2 (MR2) After the basal nociceptive responses were recorded in the tail-pinch (Al, A2 and A3) and hot-plate tests (Bl, B2 and B3), clonidine (CLN: 0.5-2.5 mg/kg, s.c.) was administered and the latency of nociceptive responses was measured every 15 over a 60-min observation period (Al, Bl) In some experiments, idazoxan (A2, B2) or MR2 (A3, B3) was administered i.p 30 mm before clonidine (1 mg/kg, s.c.) and the latency of nociceptive responses was measured as described above The numbers in parentheses are the doses administered Each point represents the mean %MPE S.E.M (n=lO-12) ‘PcO.05, *+P