Antinociceptive Mechanisms of Platycodin D Administered Intracerebroventricularly in the Mouse

• Abstract
• Introduction
• Materials and Methods
• Experimental animals
• Assessment of antinociception
• Intracerebroventricular (i. c. v.) and intrathecal (i. t.) injection
• Statistics
• Drugs
• Results
• Effects of platycodin D on tail-flick response
• Effects of MK-801 and CNQX injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered
  platycodin D
• Effects of muscimol and baclofen injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered
  platycodin D
• Effects of CCK-8s injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered platycodin D
• Effects of yohimbine, methysergide and naloxone injected i. t. on inhibition of the tail-flick response induced by i. c. v.
  administered platycodin D
• Discussion
• Acknowledgements
• References

Abstract

Platycodin D administered intracerebroventricularly (i. c. v.) showed an antinociceptive effect in a dose-dependent manner as measured by the tail-flick assay. The antinociception induced by platycodin D was maintained at least 1 h. MK-801 [(±)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], a competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a non-NMDA receptor antagonist, muscimol (a GABA_A receptor agonist), or baclofen (a GABA_B receptor antagonist), or sulfated cholecystokinin (CCK-8 s; CCK_A receptor agonist), injected i. c. v. significantly reduced the inhibition of the tail-flick response induced by platycodin D administered i. c. v. Additionally, intrathecal (i. t.) pretreatment with yohimbine (an α₂-adrenergic receptor antagonist) or methysergide (a serotonin receptor antagonist) dose-dependently attenuated inhibition of the tail-flick response induced by i. c. v. administered platycodin D. However, naloxone (an opioid receptor antagonist) did not affect the inhibition of the tail-flick response induced by platycodin D. Our results suggest that platycodin D has an antinociceptive effect when it is administered supraspinally, and supraspinal GABA_A, GABA_B, NMDA and non-NMDA receptors are involved in platycodin D-induced antinociception. Furthermore, platycodin D administered supraspinally produces antinociception by stimulating descending noradrenergic and serotonergic, but not opioidergic, pathways.

Introduction

Platycodon grandiflorum A. DC. (Campanulaceae) is a perennial herb that is distributed in East Asia including Korea, Japan and China. Its roots are edible and used in oriental traditional medicine as an antitussive or expectorant for pulmonary disease [1]. Previously, platycodin D (PD), one of several triterpene saponins, has been isolated from roots of this plant and its chemical structure determined as shown Fig. [1] [2]. As part of a phytochemical study on the roots of Platycodon grandiflorum A. DC., PD was recently isolated [3].

It has been reported that PD has some pharmacological effects, for example, stimulation of pancreatic exocrine secretion of rats which was inhibited by loxiglumide, a CCK receptor antagonist [4]. We performed a preliminary study and found that PD administered supraspinally has a strong antinociceptive effect. It is suggested that PD may act on the central nervous system. Therefore, attempts to find antinociceptive mechanisms of PD administered intracerebroventricularly (i. c. v.) were made. We and others have previously reported that various neurotransmitters such as cholecystokinin, opioids, glutamate, GABA, norepinephrine and serotonin are involved in nociceptive pathways. For example, opioids administered i. c. v. produce profound antinociception [5], Both NMDA and non-NMDA receptors are involved in opioid-induced antinociception [6], [7]. In addition, GABAergic neuron at the supraspinal level appears to play an important role in mediating antinociception induced by opioids [8]. Furthermore, the descending opioidergic, noradrenergic and serotonergic systems in the spinal cord are involved in antinociception induced by opioids or glutamate administered supraspinally [9], [10], [11]. We now report that PD administered intracerebroventricularly (i. c. v.) has a strong antinociceptive effect and PD-induced antinociception is mediated by glutamatergic-, GABAergic-receptors at supraspinal levels. CCK receptors may have an antagonistic effect against supraspinally administered PD-induced antinociception. In addition, i. c. v. administered PD produces its antinociception in part by activating the descending noradrenergic and serotonergic system.

Materials and Methods

Experimental animals

Male ICR mice weighing 23 - 25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 22 ± 0.5 °C with an alternating 12 hour light-dark cycle. Food and water were available ad libitum. Animals were used only once in all the experiments. These experiments were approved by the University of Hallym Animal Care and Use Committee. All procedures were conducted in accordance with the ”Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.

Assessment of antinociception

Antinociception was determined by the tail-flick test [12]. 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 to 5 sec. The tail-flick latencies were measured before (T₀) and after (T₁) the injection of platycodin D. The inhibition of the tail-flick response was expressed as ”percent of maximal possible effect” which was calculated as [(T₁ - T₀)/(T₂ - T₀)] × 100, where the cutoff time (T₂), which was set at 10 sec for the tail-flick test.

Intracerebroventricular (i. c. v.) and intrathecal (i. t.) injection

Intrathecal injections were made according to the procedure of Hylden and Wilcox [13] using a 25 μl Hamilton syringe with a 30 gauge needle. The i. c. v. administration followed the method described by Haley and McCormick [14]. The i. c. v. and i. t. injection volumes were 5 μl and the injection sites were verified by injecting a similar volume 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. c. v. was found to be distributed through the ventricular spaces and reached the ventral surface of the brain and the upper cervical portion of the spinal cord. The dye injected i. t. was distributed both rostrally and caudally but with short distance (about 0.5 cm) and no dye was found visually in the brain. The success rate for the injections was consistently found to be over 95 %, before the experiments were done.

Statistics

Statistical analysis was carried out by Student’s test. P values less than 0.05 were considered to be statistically significant.

Drugs

The drugs used in the present experiments were muscimol, baclofen, yohimbine, methysergide, CNQX and MK-801 (Research Biomedicals Inc., Natick, MA, USA). Sulfated cholecystokinin octapeptide (CCK-8s) was purchased from Peninsula Laboratory Inc. (Belmont, CA, USA). Platycodin D was isolated in Dr. Tae-Hee Lee’s laboratory and its purity was about 99 % [3]. CCK-8s was dissolved in sterile saline. All other drugs used for injection were dissolved 10 % DMSO solution.

Results

Effects of platycodin D on tail-flick response

Mice were injected i. c. v. with 5 μl of PD at the dose of 2 μg and the tail-flick response was measured 7.5, 15, 30, 45, and 60 min after injection. As revealed in Fig. [2] a, PD at the dose of 2 μg produced the inhibition of the tail-flick response, which reached a peak at 15 min and maintained antinociceptive level at 60 min after the injection. Various doses (from 0.5 to 2 μg) of PD dose-dependently increased inhibition of the tail-flick response (Fig. [2] b). 10 % DMSO control group did not show any effect on the basal tail-flick latency.

Effects of MK-801 and CNQX injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered
platycodin D

To determine if glutamate receptors are involved in PD-induced antinociception, various doses of MK-801 (from 0.01 to 1 μg) or CNQX (from 0.05 to 0.5 μg) were pretreated i. c. v. for 10 min and 2 μg of PD was administered i. c. v. As shown in Fig. [3], both MK-801 and CNQX significantly attenuated i. c. v. administered PD-induced inhibition of the tail-flick response in a dose-dependent manner. Either MK-801 or CNQX administered i. c. v. alone did not affect the basal tail-flick response (Fig. [3]).

Effects of muscimol and baclofen injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered
platycodin D

To determine if GABAergic receptors are involved in PD-induced antinociception, various doses muscimol (from 50 to 200 ng) or baclofen (from 2.5 to 10 ng) were pretreated i. c. v. for 10 min and then, 2 μg of PD was administered i. c. v. Pretreatment of muscimol or baclofen dose-dependently attenuated inhibition of the tail-flick response induced by PD (Fig. [4]). Both muscimol and baclofen injected i. c. v. did not affect the basal tail-flick response (Figs. [4] a and b).

Effects of CCK-8s injected i. c. v. on inhibition of the tail-flick response induced by i. c. v. administered platycodin D

To determine the role of CCK receptors involved in PD-induced antinociception, various doses of CCK-8s (from 0.05 to 0.5 ng) were pretreated i. c. v. for 10 min and 2 μg of PD was administered i. c. v. As shown in Fig. [5], CCK-8s dose-dependently attenuated i. c. v. administered PD-induced inhibition of the tail-flick response. CCK-8s administered i. c. v. alone did not affect the basal tail-flick response (Fig. [5]).

Effects of yohimbine, methysergide and naloxone injected i. t. on inhibition of the tail-flick response induced by i. c. v.
administered platycodin D

To examine whether PD produces antinociception by activating the descending pain control system, either various doses of yohimbine (from 1 to 20 μg), methysergide (1 to 20 μg) or naloxone (from 1 to 20 μg) was pretreated i. t. for 10 min and then, 2 μg of PD was administered i. c. v. The blockade of spinal α₂-adrenergic and 5-HT receptors by yohimbine and methysergide, respectively, significantly attenuated the inhibition of the tail-flick response induced by PD administered i. c. v. in a dose-dependent manner (Figs. 6a and b). However, the blockade of spinal opioidergic receptors by naloxone did not affect the inhibition of the tail-flick response induced by PD administered i. c. v. (Fig. [6] b). Yohimbine, methysergide, or naloxone administered i. t. alone did not affect the basal tail-flick response (Fig. [6]).

Discussion

In the present study, we found that platycodin D administered i. c. v. produced a profound antinociception. Its antinociceptive effect was dose-dependent and maintained for at least 60 min. Furthermore, we have demonstrated that MK-801, CNQX, muscimol, baclofen, or CCK-8 s injected i. c. v. significantly reduced the inhibition of the tail-flick response induced by platycodin D administered i. c. v. Additionally, i. t. pretreatment with yohimbine or methysergide dose-dependently attenuated inhibition of the tail-flick response induced by i. c. v. administered platycodin D. However, naloxone did not affect the inhibition of the tail-flick response induced by platycodin D.

We have previously demonstrated that glutamate receptors are involved in the production of antinociception [6], [7]. The microinjection of NMDA or glutamate into the periaqueductal gray, or glutamate into the nucleus raphe magnus produces antinociception [9]. In addition, microinjection of glutamate into the periaqueductal gray, pontine locus coeruleus/subcoeruleus, or medullary nucleus raphe magnus inhibits spinal nociceptive transmission [10] and spinal injection of an adrenoceptor antagonist also partially attenuates the antinociception produced by microinjection of glutamate in the periaqueductal gray [9], suggesting that glutamate produces antinociception by stimulating the descending pain inhibitory system. In the present study, we found that supraspinal injection of MK-801 (an NMDA receptor antagonist) or CNQX (a non-NMDA receptor antagonist) effectively attenuated antinociception induced by supraspinally administered PD. The results indicate that supraspinal NMDA and non-NMDA receptors are involved in supraspinally administered PD-induced antinociception.

Recent studies have demonstrated that gamma-aminobutyric acid (GABA) receptors are involved in the production of antinociception induced by opioids such as morphine and β-endorphin. For example, the i. c. v. administration of muscimol, a potent GABA_A receptor agonist, counteracted the antinociceptive effect of morphine and β-endorphin in rats as measured by the tail-flick method [8]. In addition, microinjection of muscimol into the periaqueductal gray attenuated morphine-induced antinociception in rats [15]. We found in the present study that muscimol (a GABA_A receptor agonist) or baclofen (a GABA_B receptor agonist) administered i. c. v. effectively attenuated antinociception induced by i. c. v. administered PD. The results suggest that supraspinal GABA_A and GABA_B receptors are involved in supraspinally administered PD-induced antinociception.

Several studies have shown that sulfated cholecystokinin octapeptide (CCK-8s) is also involved in antagonizing the antinociception induced by opioids. CCK-8s antagonizes the antinociception induced by opioid agonists [16], [17], [18]. Especially, Faris et al. [17] and Watkins et al. [18] have suggested that CCK-8s in the spinal cord may play an important role in attenuating the antinociception induced by opioids. The supraspinal injection of CCK-8s or microinjection of CCK-8s into the periaqueductal gray attenuated antinociception induced by i. c. v. administered β-endorphin- and systemically administered morphine [16], [19]. In the present study, we found that CCK-8 s injected i. c. v. effectively attenuated antinociception induced by PD administered i. c. v., suggesting that CCK-8s may have an important role for antagonizing antinociception induced by PD administered supraspinally.

The roles of descending pain inhibitory systems involved in antinociception induced by opioid administered supraspinally have been demonstrated. For example, the antinociception induced by opioids administered supraspinally is mediated in part by the activation of either spinopetal serotonergic, noradrenergic, or opioidergic system [9], [11]. This is supported by the findings that blockade of the spinal noradrenergic or serotonergic receptors by spinal injection of yohimbine, phentolamine or methysergide antagonized the antinociception induced by morphine administered supraspinally [9], [11]. In addition, we have reported that β-endorphin given supraspinally is mediated through the release of Met-enkephalin and stimulation of opioid receptors in the spinal cord. This contention is based on the findings that β-endorphin, when injected supraspinally, released Met-enkephalin from the spinal cord and the blockade of opioid receptors in the spinal cord by spinal injection of naloxone antagonized the antinociception induced by β-endorphin given supraspinally [20]. We found in the present study that spinal injection of yohimbine (an α₂-adrenergic receptor antagonist) or methysergide (a serotonergic receptor antagonist) effectively attenuated antinociception induced by supraspinally administered PD. However, spinal injection of naloxone did not affect supraspinally administered PD-induced antinociception, revealing that spinal α₂-adrenergic and serotonergic, but not opioidergic, receptors are involved in supraspinally administered PD-induced antinociception.

In conclusion, PD administered supraspinally produces profound antinociception. Its antinociception is mediated by glutamate (NMDA and non-NMDA) and GABAergic (GABA_A and GABA_B) receptors at the supraspinal level. CCK-8s has an antagonistic action against PD-induced antinociception. In addition, supraspinally administered PD produces antinociception via activating the descending spinopetal noradrenergic and serotonergic, but not opioidergic, pathway.

Acknowledgements

This research was supported by the A. R. Fund from Dongbu Hannong Chemical Co. (1995) and the Hallym Academy of Sciences at Hallym University, South Korea (2002 - 2).

References

• 1 Takagi K, Lee E B. Pharmacological studies on Platycodon grandiflorum A. DC. 3. Activities of crude platycodin on respiratory and circulatory systems and its other pharmacological activities. Yakugaku Zasshi.  J Pharm Soc Japan. 1972;  92 969-73
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  CrossrefPubMedSearch in Google Scholar
• 17 Faris P L, Komisaruk B R, Watkins L R, Mayer D J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia.  Science. 1983;  219 310-2
  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 19 Li Y, Han J S. Cholecystokinin-octapeptide antagonizes morphine analgesia in periaqueductal gray of the rat.  Brain Res. 1989;  480 105-10
  CrossrefPubMedSearch in Google Scholar
• 20 Suh H H, Collins K A, Tseng L F. Intrathecal cholecystokinin octapeptide attenuates the antinociception and release of immunoreactive Met-enkephalin induced by intraventricular beta-endorphin in the rat.  Neuropeptides. 1992;  21 131-7
  CrossrefPubMedSearch in Google Scholar

Hong-Won Suh, Ph. D., Associate professor

Department of Pharmacology and Institute of Natural Medicine

College of Medicine, Hallym University

1 Okchun-Dong, Chunchon, Kangwon-Do, 200-702, South Korea

Phone: +82-33-240-1654

Fax: +82-33-240-1652

Email: hwsuh@hallym.ac.kr

References

• 1 Takagi K, Lee E B. Pharmacological studies on Platycodon grandiflorum A. DC. 3. Activities of crude platycodin on respiratory and circulatory systems and its other pharmacological activities. Yakugaku Zasshi.  J Pharm Soc Japan. 1972;  92 969-73
  PubMedSearch in Google Scholar
• 2 Tada A, Kaneiwa Y, Shoji J, Shibata S. Studies on the saponins of the root of Platycodon grandiflorum A. De Candolle. I. Isolation and the structure of platycodin-D.  Chem Pharm Bull (Tokyo). 1975;  23 2965-72
  PubMedSearch in Google Scholar
• 3 Kim T -J, Lee S -I, Lee T -H, Ko J -S. Isolation and determination of platycodin D from platycodi radix.  Anal Sci Tech, J Kor Soc Anal Sci. 1990;  3 399-404
  PubMedSearch in Google Scholar
• 4 Arai I, Komatsu Y, Hirai Y, Shingu K, Ida Y, Yamaura H, Yamamoto T, Kuroiwa Y, Sasaki K, Taguchi S. Stimulative effects of saponin from kikyo-to, a Japanese herbal medicine, on pancreatic exocrine secretion of conscious rats.  Planta Med. 1997;  63 419-24
  PubMedSearch in Google Scholar
• 5 Suh H H, Tseng L F. Different types of opioid receptors mediating analgesia induced by morphine, DAMGO, DPDPE, DADLE and beta-endorphin in mice.  Naunyn-Schmiedeberg"s Arch of Pharmacol. 1990;  342 67-71
  PubMedSearch in Google Scholar
• 6 Suh H W, Song D K, Kim Y H, Yoo J S, Tseng L F. Differential antagonism by MK-801 against antinociception induced by opioid receptor agonists administered supraspinally in mice.  Eur J Pharmacol. 1994;  263 217-21
  CrossrefPubMedSearch in Google Scholar
• 7 Suh H W, Choi Y S, Yoo J S, Song D K, Kim Y H, Tseng L F. Non-NMDA receptor antagonist attenuates antinociception induced by morphine but not beta-endorphin, D-Pen2-D-Pen5-enkephalin, and U50, 488H administered intracerebroventricularly in mice.  Neuropeptides. 1995;  28 125-9
  CrossrefPubMedSearch in Google Scholar
• 8 Mantegazza P, Tammiso R, Vicentini L, Zambotti F, Zonta N. Muscimol antagonism of morphine analgesia in rats.  Br J Pharmacol. 1979;  67 103-7
  PubMedSearch in Google Scholar
• 9 Jensen T S, Yaksh T L. Spinal monoamine and opiate systems partly mediate the antinociceptive effects produced by glutamate at brainstem sites.  Brain Res. 1984;  321 287-97
  CrossrefPubMedSearch in Google Scholar
• 10 Jones S L, Gebhart G F. Inhibition of spinal nociceptive transmission from the midbrain, pons and medulla in the rat: activation of descending inhibition by morphine, glutamate and electrical stimulation.  Brain Res. 1988;  460 281-96
  CrossrefPubMedSearch in Google Scholar
• 11 Suh H H, Fujimoto J M, Tseng L L. Differential mechanisms mediating beta-endorphin- and morphine-induced analgesia in mice.  Eur J of Pharmacol. 1989;  168 61-70
  PubMedSearch in Google Scholar
• 12 D'Amour F E, Smith D L. A method for determining loss of pain sensation.  J Pharmacol Exp Ther. 1941;  72 74-9
  PubMedSearch in Google Scholar
• 13 Hylden J L, Wilcox G L. Intrathecal morphine in mice: a new technique.  Eur J Pharmacol. 1980;  67 313-6
  CrossrefPubMedSearch in Google Scholar
• 14 Haley T, McCormick W G. Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.  Br J Pharmacol. 1957;  12 12-5
  PubMedSearch in Google Scholar
• 15 Zambotti F, Zonta N, Parenti M, Tommasi R, Vicentini L, Conci F, Mantegazza P. Periaqueductal gray matter involvement in the muscimol-induced decrease of morphine antinociception.  Naunyn-Schmiedeberg's Arch Pharmacol. 1982;  318 368-9
  PubMedSearch in Google Scholar
• 16 Itoh S, Katsuura G, Maeda Y. Caerulein and cholecystokinin suppress beta-endorphin-induced analgesia in the rat.  Eur J Pharmacol. 1982;  80 421-5
  CrossrefPubMedSearch in Google Scholar
• 17 Faris P L, Komisaruk B R, Watkins L R, Mayer D J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia.  Science. 1983;  219 310-2
  PubMedSearch in Google Scholar
• 18 Watkins L R, Kinscheck I B, Mayer D J. Potentiation of opiate analgesia and apparent reversal of morphine tolerance by proglumide.  Science. 1984;  224 395-6
  PubMedSearch in Google Scholar
• 19 Li Y, Han J S. Cholecystokinin-octapeptide antagonizes morphine analgesia in periaqueductal gray of the rat.  Brain Res. 1989;  480 105-10
  CrossrefPubMedSearch in Google Scholar
• 20 Suh H H, Collins K A, Tseng L F. Intrathecal cholecystokinin octapeptide attenuates the antinociception and release of immunoreactive Met-enkephalin induced by intraventricular beta-endorphin in the rat.  Neuropeptides. 1992;  21 131-7
  CrossrefPubMedSearch in Google Scholar

Hong-Won Suh, Ph. D., Associate professor

Department of Pharmacology and Institute of Natural Medicine

College of Medicine, Hallym University

1 Okchun-Dong, Chunchon, Kangwon-Do, 200-702, South Korea

Phone: +82-33-240-1654

Fax: +82-33-240-1652

Email: hwsuh@hallym.ac.kr