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Antinociceptive mechanisms of Dipsacus saponin C administered intrathecally in mice
Journal of Ethnopharmacology 71 (2000) 211 – 218 www.elsevier.com/locate/jethpharm
Antinociceptive mechanisms of Dipsacus saponin C administered intrathecally in mice Hong-Won Suh a,*, Dong-Keun Song a, Sung-Oh Huh a, Kun-Ho Son b, Yung-Hi Kim a a
Department of Pharmacology, Institute of Natural Medicine, College of Medicine, Hallym Uni6ersity, 1 Okchun-Dong, Chunchon, 200 -702, Korea b Department of Food and Nutrition, Andong National Uni6ersity, Andong, 760 -749, South Korea Received 17 March 1999; received in revised form 3 October 1999; accepted 8 December 1999
Abstract Dipsacus saponin C (DSC) administered intrathecally (i.t.) showed antinociceptive effect in a dose-dependent (from 3.75 to 30 mg) manner as measured by the tail-flick assay. The antinociception induced by DSC at the dose of 30 mg reached at peak 7.5 min and almost returned to the control level after 60 min. 5-Amino-valeric acid (5-AVA, a GABAA receptor antagonist, from 1 to 20 mg) and SR 95531 (a GABAB receptor antagonist, from 0.1 to 2 ng) dose-dependently attenuated i.t. administered DSC-induced increase of the inhibition of the tail-flick response. The i.t. injection of yohimbine (an a2-adrenergic receptor antagonist, from 1 to 20 mg) and methysergide (a serotonin receptor antagonist, from 1 to 20 mg), but not naloxone (from 2 to 8 mg), significantly attenuated inhibition of the tail-flick response induced by DSC (30 mg) administered i.t. Sulfated cholecystokinin (CCK, from 0.05 to 0.5 ng) injected i.t. significantly reduced the inhibition of the tail-flick response induced by DSC (30 mg) administered i.t. Our results suggest that DSC shows an antinociceptive effect when it is administered spinally and GABAA, GABAB, a2-adrenergic and serotonin receptors located at the spinal cord level, but not opioid receptors, may be involved in DSC-induced antinociception. Furthermore, CCK may play an important role for the modulation of i.t. injected DSC-induced antinociception. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dipsacus saponin C; Antinociception; GABA; Seorotonin and noradrenergic receptors; CCK
1. Introduction Abbre6iations: 5-AVA, 5-aminovaleric acid; CCK, cholecystokinin octapeptide; GABA, gamma aminobutyric acid; i.c.v., intracerebroventricular; i.t., intrathecal; SR 95531, 2-(carboxyl-3%-propyl]-3-amino-6-paramethoxy-phenyl-pyridazinium bromide). * Corresponding author. Tel.: + 82-361-581654; fax: + 82361-581652. E-mail address: [email protected] (H.-W. Suh)
Dipsacus asper Wall (Dipsacaceae) is a perennial herb that is distributed in China. Its roots have been used in traditional medicine as analgesic, for enhancement of liver activity, as an anti-inflammatory agent, and for the treatment of fractures (Shanghai Sci. and Tech. Pub. and
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Shougakukan, 1985; Namba, 1986). Recently, several saponins and iridoid glycosides were isolated from this plant (Kouno et al., 1990; Zhang and Xue, 1991a,b). As a part of phytochemical study on the roots of Dipsacus asper, we have recently isolated a few triterpene glycosides (Jung et al., 1993a and Jung et al., 1993b). One of triterpenes was named dipsacus saponin C (DSC) as decribed in the previous reports (Jung et al., 1993b; Suh et al., 1996a). Previously, we have reported that DSC administered supraspinally exerts a strong antinociceptive effect. In the present study, attempts to find antinociceptive mechanisms of DSC administered intrathecally (i.t.) were made. We and others have previously reported that various neurotransmitters such as cholecystokinin, opioids, GABA, norepinephrine and serotonin are involved in nociceptive pathway. For example, opioids administered i.t. produce profound antinociception (Yaksh, 1983; Yaksh and Noueihed, 1985; Suh et al., 1996b). In addition, GABAergic neuron at supraspinal and spinal cord levels appears to play an important role in mediating antinociception (Mantegazza et al., 1979; Zonta et al., 1981; Aran and Hammond, 1991; Dirig and Yaksh, 1995; Nadeson et al., 1996). Furthermore, the noradrenergic and serotonnin systems in the spinal cord are involved in antinociception (Hoehn et al., 1988; Xu et al., 1994; Tseng and Collins, 1996; Bardin et al., 1997; Yang et al., 1998). We now report that DSC administered intrathecally also possesses a strong antinociceptive effect and DSCinduced antinociception may be mediated by GABAergic, serotonergic and a2-adrenergic receptors at the spinal cord level. CCK receptors may have an antagonistic effect against spinally administered DSC-induced antinociception.
2. Material and methods Dipsacus saponin C was isolated in the laboratory of Dr Kun-Ho Son (Jung et al., 1993b).
all the experiments. Animals were housed five per cage in a room maintained at 229 0.5°C with an alternating 12 h light–dark cycle. Food and water were available ad libitum. Animals were used only once in all the experiments.
2.2. Assessment of antinociception Antinociception was determined by the tail-flick test (D’Amour and Smith, 1941). For the measurement of the latency of the tail-flick response, mice were gently held with one hand with the tail positioned in the apparatus (EMDIE Instrument Co., Maidens, VA, Model TF6) for radiant heat stimulation. The tail-flick response was elicited by applying radiant heat on the dorsal surface of the tail. The intensity of heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail within 3–5 s. The tail-flick latencies were measured before (T0) and after (T1) the injection of opioid agonists. The inhibition of the tailflick response was expressed as percent maximum possible effect (% MPE) which was calculated as [(T1 − T0)/(T2 − T0)]× 100, where the cutoff time (T2) which was set at 10 s for the tail-flick test.
2.3. Intrathecal (i.t.) injection Intrathecal injections were made according to the procedure of Hylden and Wilcox (1980) using a 25 ml Hamilton syringe with a 30 gauge needle. The i.t. injection volumes were 5 ml and the injection sites were verified by injecting a similar vol. of 1% methylene blue solution and determining the distribution of the injected dye in the ventricular space or in the spinal cord. The dye injected i.t. was distributed over a short distance (about 0.5 cm) both rostrally and caudally and no dye was found in the brain. The success rate for the injections was consistently found to be over 95%, before the experiments were done.
2.4. Statistics 2.1. Experimental animals Male ICR mice weighing 23 – 25 g were used for
Statistical analysis was carried out by one-way analysis of variance (ANOVA) with post hoc
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multiple comparisons test. P-values less than 0.05 were considered to indicate statistical significance.
2.5. Drugs The drugs used in the present experiments were 5-aminovaleric acid, SR 95531, yohimbine hydrochloride, methysergide maleate, and naloxone (Research Biomedicals, Natick, MA). Cholecystokinin octapeptide (CCK) was purchased from Peninsula Laboratory (Belmont, CA). 5-aminovaleric acid, SR 95531, CCK, naloxone were dis-
Fig. 2. Effects of 5-aminovaleric acid (5-AVA) (a) and SR 95531 (b) injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Saline (5 ml), 5-AVA (from 1 to 20 mg) or SR 95531 (from 0.1 to 2 ng) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * PB0.05 compared to the group of mice treated with saline plus DSC.
solved in sterile saline (0.9% NaCl solution). Yohimbine, methysergide and DSC were dissolved in dimethyl sulfoxide (20%). Fig. 1. Antinociceptive effect of DSC: time (a)- and dose (b)-dependent experiment. (a) Thirty micrograms of DSC were administered intrathecally (i.t.) and the tail-flick response was measured at 7.5, 15, 30, 45 and 60 min after injection. (b) Various doses (3.75, 7.5, 15 and 30 mg) of DSC was administered i.t. and the tail-flick response was measured 7.5 min after injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10.
3. Results
3.1. Effects of DSC on tail-flick response Mice were injected i.t. with 5 ml of DSC at the dose of 30 mg and the tail-flick response was
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measured 7.5, 15, 30, 45, and 60 min after injection. As revealed in Fig. 1a, DSC at the dose of 30 mg produced the inhibition of the tail-flick response, which reached at peak 7.5 min and its antinociceptive effect was almost returned to the control level 60 min after the injection. Various doses (3.75–30 mg) of DSC dose-dependently produced inhibition of the tail-flick response (Fig. 1b). In addition, mice administered i.t. with DSC
Fig. 4. Effects of CCK injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Saline or CCK (from 0.05 to 0.5 ng) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * P B0.05 compared to the group of mice treated with DSC.
at higher doses (i.e. higher than 30 mg) showed an excitatory behavior.
3.2. Effects of 5 -AVA and SR 95531 injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC
Fig. 3. Effects of yohimbine (a) and methysergide (b) injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Vehicle (20% DMSO), yohimbine (from 1 to 20 mg) or methysergide (from 1 to 20 mg) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * P B 0.05 compared to the group of mice treated with vehicle plus DSC.
To determine if the spinal GABAergic receptors are involved in DSC-induced antinociception, various doses of 5-AVA (from 1 to 20 mg) or SR 95531 (from 0.1 to 2 ng) were pretreated i.c.v. 10 min before i.t. injection of DSC (30 mg). As shown in Fig. 2, both 5-AVA and SR 95531 attenuated i.t. administered DSC-induced inhibition of the tail-flick response in a dose-dependent manner. Either MK-801 plus vehicle, or CNQX plus vehicle administered i.t. did not affect the basal tailflick response (data not shown).
3.3. Effects of yohimbine and methysergide injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC To determine if DSC produces antinociception by stimulating monoaminergic systems in the spinal cord, various doses of yohimbine (from 1
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to 20 mg), or methysergide (from 1 to 20 mg) was pretreated i.t. for 10 min and then, 30 mg of DSC was administered i.t. The blockade of spinal a2-adrenergic and serotonergic receptors by yohimbine and methysergide, respectively, dose-dependently attenuated the inhibition of the tail-flick response induced by DSC administered i.t. (Fig. 3). Yohimbine plus vehicle, methysergide plus vehicle, or naloxone plus vehicle administered i.t. did not affect the basal tail-flick response (data not shown).
3.4. Effects of CCK and naloxone injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC To determine the role of CCK receptors located in the spinal cord involved in DSC-induced antinociception, various doses (from 0.05 to 0.5 ng) of CCK were pretreated i.t. 10 min before i.t. administration of DSC (30 mg). As shown in Fig. 4, CCK dose-dependently attenuated i.t. administered DSC-induced inhibition of the tail-flick response. CCK plus vehicle administered i.t. did not affect the basal tail-flick response (data not shown). Furthermore, to determine if the spinal opioid receptors are involved in DSC-induced antinociception, various doses (from 2 to 8 mg) of naloxone were pretreated i.t. 10 min before i.t. administration of DSC (30 mg). Naloxone plus vehicle (20% DMSO), which did not affect the basal tail-flick response at the dose used, did not affect the inhibition of the tail-flick response induced by DSC (data not shown).
4. Discussion In the present study, we found that DSC administered i.t. produces antinociception and its antinociceptive effect is dose-dependent. It is well known that opioids produce strong antinociception when they are administered supraspinally or spinally (Porreca et al., 1987; Suh and Tseng, 1988). The antinociception induced by DSC administered spinally may not be mediated by opioid receptors. This contention is
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supported by the finding that the blockade of spinal opioid receptors by naloxone, an opioid receptor antagonist, did not affect the antinociception induced by DSC administered i.t. The result of the present study partially in line with our presvious study that supraspinally injected naloxone does not affect the antinociception induced by supraspinally administered DSC (Suh et al., 1996a). Recent studies have demonstrated that a gamma amino butyric acid (GABA) receptors are involved in the production of antinociception induced by opioids. For example, the supraspinal administration of muscimol, a potent GABAA receptor agonist, counteracted the antinociceptive effect of morphine and b-endorphin in rats or mice (Mantegazza et al., 1979; Zonta et al., 1981; Zambotti et al., 1982; Suh et al., 1995). In addition, in the spinal cord level, muscimol or baclofen produces the antinociception (Mantegazza et al., 1979; Zonta et al., 1981; Aran and Hammond, 1991; Dirig and Yaksh, 1995; Nadeson et al., 1996). We found in the present study that 5-AVA (a GABAA receptor antagonist) or SR95531 (a GABAB receptor antagonist) administered i.t. effectively attenuated antinociception induced by i.t. administered DSC. The results suggest that both GABAA and GABAB receptors located at the spinal cord may be involved in spinally administered DSC-induced antinociception. The roles of the spinal monoamidergic systems in the regulation of the nociception have been demonstrated in several studies. For example, i.t. injection of serotonin or norepinephrine produces antinociception (Kuraishi et al., 1985; Post et al., 1985; Zhang et al., 1995; Tseng and Collins, 1996). Furthremore, the antinociception induced by opioids administered supraspinally is mediated in part by the activation of either spinopetal serotonergic, noradrenergic receptors (Yaksh, 1979; Jensen and Yaksh, 1984; Wigdor and Wilcox, 1987; Suh et al., 1989). We found in the present study that spinal injection of yohimbine (an a2-adrenergic receptor antagonist) or methysergide (a serotonin receptor antagonist) effectively attenuated antinociception induced by spinally administered DSC. Furthermore, in a
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previous study, we found that i.t. injection of yohimbine or methysergide attenuates i.c.v. administered DSC-induced antinociception (Suh et al., 1996b). Taken together, it is suggested that spinal a2-adrenergic and serotonergic receptors may be involved in supraspinally or spinally administered DSC-induced antinociception. Several studies have shown that cholecystokinin octapeptide (CCK) is also involved in antagonizing the antinociception induced by opioids. CCK antagonizes the antinociception induced by opioid agonists (Itoh et al., 1982; Faris et al., 1983; O’Neill et al., 1989). Especially, Faris et al. (1983) and Watkins et al. (1984) have suggested that CCK in the spinal cord may play an important role in attenuating the antinociception induced by opioids. This view is supported by the findings that CCK injected spinally antagonizes spinally injected morphine-induced and naloxone-reversible stress-induced antinociception, indicating that CCK in the spinal cord antagonizes both exogenous and endogenous opioid induced antinociception (Watkins et al., 1985a,b). The supraspinal injection of CCK or microinjection of CCK into the periaqueductal gray attenuated antinociception induced by intracerebroventricularlly (i.c.v.) administered bendorphin- and systemically administered morphine (Itoh et al., 1982; Li and Han, 1989). In our previous study, we found that CCK injected i.c.v. effectively attenuates the antinociception induced by DSC administered i.c.v, suggesting that CCK may have an important role for antagonizing antinociception induced by DSC administered supraspinally. In addition, in the present study, we found that i.t. injected CCK antagonizes the antinociception induced by DSC administered i.t., suggesting that CCK located in the spinal cord may have an important role for antagonizing antinociception induced by DSC administered spinally. Furthermore, from the finding of the fact that i.t. administered naloxone failed to antagonize the antinociception induced by i.t. administered DSC, it is speculated that the antagonistic effect of CCK is not limited to opioids-induced antinociception.
In conclusion, DSC administered spinally produces antinociception. Its antinociception appears to be mediated by GABAergic, serotonergic, and noradrenergic receptors at the spinal cord level. Furthermore, CCK has an antagonistic action against DSC-induced antinociception at the spinal cord level.
Acknowledgements This work was supported by the Research Fund from Ministry of Science and Technology in Korea.
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Antinociceptive mechanisms of Dipsacus saponin C administered intrathecally in mice Hong-Won Suh a,*, Dong-Keun Song a, Sung-Oh Huh a, Kun-Ho Son b, Yung-Hi Kim a a
Department of Pharmacology, Institute of Natural Medicine, College of Medicine, Hallym Uni6ersity, 1 Okchun-Dong, Chunchon, 200 -702, Korea b Department of Food and Nutrition, Andong National Uni6ersity, Andong, 760 -749, South Korea Received 17 March 1999; received in revised form 3 October 1999; accepted 8 December 1999
Abstract Dipsacus saponin C (DSC) administered intrathecally (i.t.) showed antinociceptive effect in a dose-dependent (from 3.75 to 30 mg) manner as measured by the tail-flick assay. The antinociception induced by DSC at the dose of 30 mg reached at peak 7.5 min and almost returned to the control level after 60 min. 5-Amino-valeric acid (5-AVA, a GABAA receptor antagonist, from 1 to 20 mg) and SR 95531 (a GABAB receptor antagonist, from 0.1 to 2 ng) dose-dependently attenuated i.t. administered DSC-induced increase of the inhibition of the tail-flick response. The i.t. injection of yohimbine (an a2-adrenergic receptor antagonist, from 1 to 20 mg) and methysergide (a serotonin receptor antagonist, from 1 to 20 mg), but not naloxone (from 2 to 8 mg), significantly attenuated inhibition of the tail-flick response induced by DSC (30 mg) administered i.t. Sulfated cholecystokinin (CCK, from 0.05 to 0.5 ng) injected i.t. significantly reduced the inhibition of the tail-flick response induced by DSC (30 mg) administered i.t. Our results suggest that DSC shows an antinociceptive effect when it is administered spinally and GABAA, GABAB, a2-adrenergic and serotonin receptors located at the spinal cord level, but not opioid receptors, may be involved in DSC-induced antinociception. Furthermore, CCK may play an important role for the modulation of i.t. injected DSC-induced antinociception. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dipsacus saponin C; Antinociception; GABA; Seorotonin and noradrenergic receptors; CCK
1. Introduction Abbre6iations: 5-AVA, 5-aminovaleric acid; CCK, cholecystokinin octapeptide; GABA, gamma aminobutyric acid; i.c.v., intracerebroventricular; i.t., intrathecal; SR 95531, 2-(carboxyl-3%-propyl]-3-amino-6-paramethoxy-phenyl-pyridazinium bromide). * Corresponding author. Tel.: + 82-361-581654; fax: + 82361-581652. E-mail address: [email protected] (H.-W. Suh)
Dipsacus asper Wall (Dipsacaceae) is a perennial herb that is distributed in China. Its roots have been used in traditional medicine as analgesic, for enhancement of liver activity, as an anti-inflammatory agent, and for the treatment of fractures (Shanghai Sci. and Tech. Pub. and
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 2 0 4 - 4
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Shougakukan, 1985; Namba, 1986). Recently, several saponins and iridoid glycosides were isolated from this plant (Kouno et al., 1990; Zhang and Xue, 1991a,b). As a part of phytochemical study on the roots of Dipsacus asper, we have recently isolated a few triterpene glycosides (Jung et al., 1993a and Jung et al., 1993b). One of triterpenes was named dipsacus saponin C (DSC) as decribed in the previous reports (Jung et al., 1993b; Suh et al., 1996a). Previously, we have reported that DSC administered supraspinally exerts a strong antinociceptive effect. In the present study, attempts to find antinociceptive mechanisms of DSC administered intrathecally (i.t.) were made. We and others have previously reported that various neurotransmitters such as cholecystokinin, opioids, GABA, norepinephrine and serotonin are involved in nociceptive pathway. For example, opioids administered i.t. produce profound antinociception (Yaksh, 1983; Yaksh and Noueihed, 1985; Suh et al., 1996b). In addition, GABAergic neuron at supraspinal and spinal cord levels appears to play an important role in mediating antinociception (Mantegazza et al., 1979; Zonta et al., 1981; Aran and Hammond, 1991; Dirig and Yaksh, 1995; Nadeson et al., 1996). Furthermore, the noradrenergic and serotonnin systems in the spinal cord are involved in antinociception (Hoehn et al., 1988; Xu et al., 1994; Tseng and Collins, 1996; Bardin et al., 1997; Yang et al., 1998). We now report that DSC administered intrathecally also possesses a strong antinociceptive effect and DSCinduced antinociception may be mediated by GABAergic, serotonergic and a2-adrenergic receptors at the spinal cord level. CCK receptors may have an antagonistic effect against spinally administered DSC-induced antinociception.
2. Material and methods Dipsacus saponin C was isolated in the laboratory of Dr Kun-Ho Son (Jung et al., 1993b).
all the experiments. Animals were housed five per cage in a room maintained at 229 0.5°C with an alternating 12 h light–dark cycle. Food and water were available ad libitum. Animals were used only once in all the experiments.
2.2. Assessment of antinociception Antinociception was determined by the tail-flick test (D’Amour and Smith, 1941). For the measurement of the latency of the tail-flick response, mice were gently held with one hand with the tail positioned in the apparatus (EMDIE Instrument Co., Maidens, VA, Model TF6) for radiant heat stimulation. The tail-flick response was elicited by applying radiant heat on the dorsal surface of the tail. The intensity of heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail within 3–5 s. The tail-flick latencies were measured before (T0) and after (T1) the injection of opioid agonists. The inhibition of the tailflick response was expressed as percent maximum possible effect (% MPE) which was calculated as [(T1 − T0)/(T2 − T0)]× 100, where the cutoff time (T2) which was set at 10 s for the tail-flick test.
2.3. Intrathecal (i.t.) injection Intrathecal injections were made according to the procedure of Hylden and Wilcox (1980) using a 25 ml Hamilton syringe with a 30 gauge needle. The i.t. injection volumes were 5 ml and the injection sites were verified by injecting a similar vol. of 1% methylene blue solution and determining the distribution of the injected dye in the ventricular space or in the spinal cord. The dye injected i.t. was distributed over a short distance (about 0.5 cm) both rostrally and caudally and no dye was found in the brain. The success rate for the injections was consistently found to be over 95%, before the experiments were done.
2.4. Statistics 2.1. Experimental animals Male ICR mice weighing 23 – 25 g were used for
Statistical analysis was carried out by one-way analysis of variance (ANOVA) with post hoc
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multiple comparisons test. P-values less than 0.05 were considered to indicate statistical significance.
2.5. Drugs The drugs used in the present experiments were 5-aminovaleric acid, SR 95531, yohimbine hydrochloride, methysergide maleate, and naloxone (Research Biomedicals, Natick, MA). Cholecystokinin octapeptide (CCK) was purchased from Peninsula Laboratory (Belmont, CA). 5-aminovaleric acid, SR 95531, CCK, naloxone were dis-
Fig. 2. Effects of 5-aminovaleric acid (5-AVA) (a) and SR 95531 (b) injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Saline (5 ml), 5-AVA (from 1 to 20 mg) or SR 95531 (from 0.1 to 2 ng) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * PB0.05 compared to the group of mice treated with saline plus DSC.
solved in sterile saline (0.9% NaCl solution). Yohimbine, methysergide and DSC were dissolved in dimethyl sulfoxide (20%). Fig. 1. Antinociceptive effect of DSC: time (a)- and dose (b)-dependent experiment. (a) Thirty micrograms of DSC were administered intrathecally (i.t.) and the tail-flick response was measured at 7.5, 15, 30, 45 and 60 min after injection. (b) Various doses (3.75, 7.5, 15 and 30 mg) of DSC was administered i.t. and the tail-flick response was measured 7.5 min after injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10.
3. Results
3.1. Effects of DSC on tail-flick response Mice were injected i.t. with 5 ml of DSC at the dose of 30 mg and the tail-flick response was
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measured 7.5, 15, 30, 45, and 60 min after injection. As revealed in Fig. 1a, DSC at the dose of 30 mg produced the inhibition of the tail-flick response, which reached at peak 7.5 min and its antinociceptive effect was almost returned to the control level 60 min after the injection. Various doses (3.75–30 mg) of DSC dose-dependently produced inhibition of the tail-flick response (Fig. 1b). In addition, mice administered i.t. with DSC
Fig. 4. Effects of CCK injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Saline or CCK (from 0.05 to 0.5 ng) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * P B0.05 compared to the group of mice treated with DSC.
at higher doses (i.e. higher than 30 mg) showed an excitatory behavior.
3.2. Effects of 5 -AVA and SR 95531 injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC
Fig. 3. Effects of yohimbine (a) and methysergide (b) injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DSC administered i.t. Vehicle (20% DMSO), yohimbine (from 1 to 20 mg) or methysergide (from 1 to 20 mg) was pretreated i.t. 10 min before i.t. administration of DSC (30 mg) or vehicle. The tail-flick response was measured 7.5 min after DSC or vehicle injection. The vertical bars denote the standard error of the mean. The number of animal used for each group was eight to 10. * P B 0.05 compared to the group of mice treated with vehicle plus DSC.
To determine if the spinal GABAergic receptors are involved in DSC-induced antinociception, various doses of 5-AVA (from 1 to 20 mg) or SR 95531 (from 0.1 to 2 ng) were pretreated i.c.v. 10 min before i.t. injection of DSC (30 mg). As shown in Fig. 2, both 5-AVA and SR 95531 attenuated i.t. administered DSC-induced inhibition of the tail-flick response in a dose-dependent manner. Either MK-801 plus vehicle, or CNQX plus vehicle administered i.t. did not affect the basal tailflick response (data not shown).
3.3. Effects of yohimbine and methysergide injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC To determine if DSC produces antinociception by stimulating monoaminergic systems in the spinal cord, various doses of yohimbine (from 1
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to 20 mg), or methysergide (from 1 to 20 mg) was pretreated i.t. for 10 min and then, 30 mg of DSC was administered i.t. The blockade of spinal a2-adrenergic and serotonergic receptors by yohimbine and methysergide, respectively, dose-dependently attenuated the inhibition of the tail-flick response induced by DSC administered i.t. (Fig. 3). Yohimbine plus vehicle, methysergide plus vehicle, or naloxone plus vehicle administered i.t. did not affect the basal tail-flick response (data not shown).
3.4. Effects of CCK and naloxone injected i.t. on inhibition of the tail-flick response induced by i.t. administered DSC To determine the role of CCK receptors located in the spinal cord involved in DSC-induced antinociception, various doses (from 0.05 to 0.5 ng) of CCK were pretreated i.t. 10 min before i.t. administration of DSC (30 mg). As shown in Fig. 4, CCK dose-dependently attenuated i.t. administered DSC-induced inhibition of the tail-flick response. CCK plus vehicle administered i.t. did not affect the basal tail-flick response (data not shown). Furthermore, to determine if the spinal opioid receptors are involved in DSC-induced antinociception, various doses (from 2 to 8 mg) of naloxone were pretreated i.t. 10 min before i.t. administration of DSC (30 mg). Naloxone plus vehicle (20% DMSO), which did not affect the basal tail-flick response at the dose used, did not affect the inhibition of the tail-flick response induced by DSC (data not shown).
4. Discussion In the present study, we found that DSC administered i.t. produces antinociception and its antinociceptive effect is dose-dependent. It is well known that opioids produce strong antinociception when they are administered supraspinally or spinally (Porreca et al., 1987; Suh and Tseng, 1988). The antinociception induced by DSC administered spinally may not be mediated by opioid receptors. This contention is
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supported by the finding that the blockade of spinal opioid receptors by naloxone, an opioid receptor antagonist, did not affect the antinociception induced by DSC administered i.t. The result of the present study partially in line with our presvious study that supraspinally injected naloxone does not affect the antinociception induced by supraspinally administered DSC (Suh et al., 1996a). Recent studies have demonstrated that a gamma amino butyric acid (GABA) receptors are involved in the production of antinociception induced by opioids. For example, the supraspinal administration of muscimol, a potent GABAA receptor agonist, counteracted the antinociceptive effect of morphine and b-endorphin in rats or mice (Mantegazza et al., 1979; Zonta et al., 1981; Zambotti et al., 1982; Suh et al., 1995). In addition, in the spinal cord level, muscimol or baclofen produces the antinociception (Mantegazza et al., 1979; Zonta et al., 1981; Aran and Hammond, 1991; Dirig and Yaksh, 1995; Nadeson et al., 1996). We found in the present study that 5-AVA (a GABAA receptor antagonist) or SR95531 (a GABAB receptor antagonist) administered i.t. effectively attenuated antinociception induced by i.t. administered DSC. The results suggest that both GABAA and GABAB receptors located at the spinal cord may be involved in spinally administered DSC-induced antinociception. The roles of the spinal monoamidergic systems in the regulation of the nociception have been demonstrated in several studies. For example, i.t. injection of serotonin or norepinephrine produces antinociception (Kuraishi et al., 1985; Post et al., 1985; Zhang et al., 1995; Tseng and Collins, 1996). Furthremore, the antinociception induced by opioids administered supraspinally is mediated in part by the activation of either spinopetal serotonergic, noradrenergic receptors (Yaksh, 1979; Jensen and Yaksh, 1984; Wigdor and Wilcox, 1987; Suh et al., 1989). We found in the present study that spinal injection of yohimbine (an a2-adrenergic receptor antagonist) or methysergide (a serotonin receptor antagonist) effectively attenuated antinociception induced by spinally administered DSC. Furthermore, in a
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previous study, we found that i.t. injection of yohimbine or methysergide attenuates i.c.v. administered DSC-induced antinociception (Suh et al., 1996b). Taken together, it is suggested that spinal a2-adrenergic and serotonergic receptors may be involved in supraspinally or spinally administered DSC-induced antinociception. Several studies have shown that cholecystokinin octapeptide (CCK) is also involved in antagonizing the antinociception induced by opioids. CCK antagonizes the antinociception induced by opioid agonists (Itoh et al., 1982; Faris et al., 1983; O’Neill et al., 1989). Especially, Faris et al. (1983) and Watkins et al. (1984) have suggested that CCK in the spinal cord may play an important role in attenuating the antinociception induced by opioids. This view is supported by the findings that CCK injected spinally antagonizes spinally injected morphine-induced and naloxone-reversible stress-induced antinociception, indicating that CCK in the spinal cord antagonizes both exogenous and endogenous opioid induced antinociception (Watkins et al., 1985a,b). The supraspinal injection of CCK or microinjection of CCK into the periaqueductal gray attenuated antinociception induced by intracerebroventricularlly (i.c.v.) administered bendorphin- and systemically administered morphine (Itoh et al., 1982; Li and Han, 1989). In our previous study, we found that CCK injected i.c.v. effectively attenuates the antinociception induced by DSC administered i.c.v, suggesting that CCK may have an important role for antagonizing antinociception induced by DSC administered supraspinally. In addition, in the present study, we found that i.t. injected CCK antagonizes the antinociception induced by DSC administered i.t., suggesting that CCK located in the spinal cord may have an important role for antagonizing antinociception induced by DSC administered spinally. Furthermore, from the finding of the fact that i.t. administered naloxone failed to antagonize the antinociception induced by i.t. administered DSC, it is speculated that the antagonistic effect of CCK is not limited to opioids-induced antinociception.
In conclusion, DSC administered spinally produces antinociception. Its antinociception appears to be mediated by GABAergic, serotonergic, and noradrenergic receptors at the spinal cord level. Furthermore, CCK has an antagonistic action against DSC-induced antinociception at the spinal cord level.
Acknowledgements This work was supported by the Research Fund from Ministry of Science and Technology in Korea.
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