Korean J Physiol Pharmacol.  2013 Apr;17(2):127-132. 10.4196/kjpp.2013.17.2.127.

Inhibitory Effects of Ginsenoside Metabolites, Compound K and Protopanaxatriol, on GABAC Receptor-Mediated Ion Currents

• Lee BH
• Hwang SH
• Choi SH
• Kim HJ
• Lee JH
• Lee SM
• Ahn YG
• Nah SY

• KMID: 1429383
• DOI: http://doi.org/10.4196/kjpp.2013.17.2.127

Abstract

Ginsenosides, one of the active ingredients of Panax ginseng, show various pharmacological and physiological effects, and they are converted into compound K (CK) or protopanaxatriol (M4) by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. The gamma-aminobutyric acid receptorC (GABAC) is primarily expressed in retinal bipolar cells and several regions of the brain. However, little is known of the effects of ginsenoside metabolites on GABAC receptor channel activity. In the present study, we examined the effects of CK and M4 on the activity of human recombinant GABAC receptor (rho1) channels expressed in Xenopus oocytes by using a 2-electrode voltage clamp technique. In oocytes expressing GABAC receptor cRNA, we found that CK or M4 alone had no effect in oocytes. However, co-application of either CK or M4 with GABA inhibited the GABA-induced inward peak current (IGABA). Interestingly, pre-application of M4 inhibited IGABA more potently than CK in a dose-dependent and reversible manner. The half-inhibitory concentration (IC50) values of CK and M4 were 52.1+/-2.3 and 45.7+/-3.9 microM, respectively. Inhibition of IGABA by CK and M4 was voltage-independent and non-competitive. This study implies that ginsenoside metabolites may regulate GABAC receptor channel activity in the brain, including in the eyes.

Keyword

GABAC receptor; Ginsenoside metabolites; Panax ginseng; Xenopus oocytes

MeSH Terms

Brain
Eye
gamma-Aminobutyric Acid
Ginsenosides
Humans
Oocytes
Panax
Retinal Bipolar Cells
RNA, Complementary
Sapogenins
Xenopus
Ginsenosides
RNA, Complementary
Sapogenins
gamma-Aminobutyric Acid

Figure

• 

  Fig. 1 Chemical structure of the ginsenoside metabolites CK and M4 (A) and their effect on oocytes expressing GABAC receptors (B). CK and M4 had no effect on IGABA in oocytes expressing GABAC receptors.

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  Fig. 2 Effect of CK and M4 on IGABA in oocytes expressing the ρ1 GABAC receptor. (A) GABA (2 µM) was first applied and then was co- or pre-treated with CK (100 µM). Co-treatment of CK with GABA and pre-treatment of CK before GABA application inhibited IGABA in oocytes expressing ρ1 GABAC receptors. (B) GABA (2 µM) was first applied and then co- or pre-treated with M4 (100 µM). Co-treatment of M4 with GABA and pre-treatment of M4 before GABA application inhibited IGABA in oocytes expressing ρ1 GABAC receptors. The resting membrane potential of oocytes was approximately -35 mV and oocytes were voltage clamped at a holding potential of -80 mV prior to drug application. Traces are representative of 8~12 separate oocytes from 3 different frogs. (C) Summary of percent inhibition by CK and M4 of IGABA calculated from the average of the peak inward current elicited by GABA alone before CK and M4 and the peak inward current elicited by GABA alone after co- and pre-treatment of CK and M4 with GABA. Each point represents the mean±S.E.M. (n=9~12 from 3 different frogs). *p <0.05 as compared to co-treatment of CK and M4, and #p<0.05 as compared to CK.

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  Fig. 3 Concentration-dependent effects of CK and M4 on IGABA in oocytes expressing ρ1 GABAC receptors. (A, B) The trace shows that CK and M4 inhibited the currents elicited by GABA (GABA; 2 µM) in a dose-dependent manner. (C) Percent inhibition by CK and M4 of IGABA was calculated from the average of the peak inward current elicited by GABA alone before CK and M4 and the peak inward current elicited by GABA alone after pre-treatment of CK and M4 before GABA. The continuous line shows the curve fitted according to the equation. Each point represents the mean±S.E.M. (n=9~12 from 3 different frogs). *p<0.005 as compared to CK.

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  Fig. 4 Voltage-independent inhibition by CK and M4. (A) GABA (2 µM) was first applied and pre-treatment of CK (100 µM) or M4 (100 µM) before GABA application inhibited IGABA in oocytes expressing ρ1 GABAC receptors. The inhibitory degrees were very similar at -30 mV and -120 mV of membrane potential. (B) Voltage-independent inhibition of IGABA in GABAC receptors by CK and M4. Values were obtained from the receptors in the absence or presence of 100 µM CK and M4 at the indicated membrane holding potentials. The observed effects of CK and M4 were correlated with membrane potential. No significant effects were noted for CK (r2=0.28, p>0.31) and M4 (r2=0.10, p>0.44).

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  Fig. 5 Current-voltage relationships and concentration-dependent effects of GABA on CK- and M4-mediated inhibition of IGABA. (A) Current-voltage relationships of IGABA inhibition by CK and M4 in GABAC receptors. Representative current-voltage relationships were obtained using voltage ramps of -100 to +40 mV for 300 ms at a holding potential of -80 mV. Voltage steps were applied before and after application of 2 µM GABA in the absence or presence of 100 µM CK and M4. (B) Concentration-response relationships for GABA in GABAC receptors with GABA applied (0.3~30 µM) alone or with GABA plus pre-treatment of 100 µM CK and M4 before GABA application. IGABA of oocytes expressing the GABAC receptors was measured using the indicated concentration of GABA in the absence (□) or presence (○) of 100 µM CK or presence (△) of 100 µM M4. Oocytes were voltage-clamped at a holding potential of -80 mV. Each point represents the mean±S.E.M. (n=8~12/group).

Reference

1. Nah SY. Ginseng: recent advances and trends. Korean J Ginseng Sci. 1997; 21:1–12.

2. Nah SY, Kim DH, Rhim H. Ginsenosides: are any of them candidates for drugs acting on the central nervous system? CNS Drug Rev. 2007; 13:381–404. PMID: 18078425.

3. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol. 1999; 58:1685–1693. PMID: 10571242.

4. Hasegawa H, Sung JH, Matsumiya S, Uchiyama M. Main ginseng saponin metabolites formed by intestinal bacteria. Planta Med. 1996; 62:453–457. PMID: 8923812.

5. Kanaoka M, Akao T, Kobashi K. Metabolism of ginseng saponins, ginsenosides by human intestinal flora. J Trad Med. 1994; 11:241–245.

6. Karikura M, Miyase T, Tanizawa H, Taniyama T, Takino Y. Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VI. The decomposition products of ginsenoside Rb2 in the stomach of rats. Chem Pharm Bull (Tokyo). 1991; 39:400–404. PMID: 2054864.

7. Hasegawa H, Suzuki R, Nagaoka T, Tezuka Y, Kadota S, Saiki I. Prevention of growth and metastasis of murine melanoma through enhanced natural-killer cytotoxicity by fatty acid-conjugate of protopanaxatriol. Biol Pharm Bull. 2002; 25:861–866. PMID: 12132658.

8. Wakabayashi C, Murakami K, Hasegawa H, Murata J, Saiki I. An intestinal bacterial metabolite of ginseng protopanaxadiol saponins has the ability to induce apoptosis in tumor cells. Biochem Biophys Res Commun. 1998; 246:725–730. PMID: 9618279.

9. Bormann J. The 'ABC' of GABA receptors. Trends Pharmacol Sci. 2000; 21:16–19. PMID: 10637650.

10. Chebib M, Johnston GA. GABA-Activated ligand gated ion channels: medicinal chemistry and molecular biology. J Med Chem. 2000; 43:1427–1447. PMID: 10780899.

11. Zhang D, Pan ZH, Awobuluyi M, Lipton SA. Structure and function of GABA_C receptors: a comparison of native versus recombinant receptors. Trends Pharmacol Sci. 2001; 22:121–132. PMID: 11239575.

12. Wässle H, Koulen P, Brandstätter JH, Fletcher EL, Becker CM. Glycine and GABA receptors in the mammalian retina. Vision Res. 1998; 38:1411–1430. PMID: 9667008.

13. McCall MA, Lukasiewicz PD, Gregg RG, Peachey NS. Elimination of the rho1 subunit abolishes GABA_C receptor expression and alters visual processing in the mouse retina. J Neurosci. 2002; 22:4163–4174. PMID: 12019334.

14. Drew CA, Johnston GA, Weatherby RP. Bicuculline-insensitive GABA receptors: studies on the binding of (-)-baclofen to rat cerebellar membranes. Neurosci Lett. 1984; 52:317–321. PMID: 6097844.

15. Strata F, Cherubini E. Transient expression of a novel type of GABA response in rat CA3 hippocampal neurones during development. J Physiol. 1994; 480:493–503. PMID: 7869263.

16. Johnston GA, Chebib M, Hanrahan JR, Mewett KN. GABA_C receptors as drug targets. Curr Drug Targets CNS Neurol Disord. 2003; 2:260–268. PMID: 12871036.

17. Choi SE, Choi S, Lee JH, Whiting PJ, Lee SM, Nah SY. Effects of ginsenosides on GABA_A receptor channels expressed in Xenopus oocytes. Arch Pharm Res. 2003; 26:28–33. PMID: 12568354.

18. Lee BH, Choi SH, Shin TJ, Hwang SH, Kang J, Kim HJ, Kim BJ, Nah SY. Effects of ginsenoside metabolites on GABA_A receptor-mediated ion currents. J Ginseng Res. 2012; 36:55–60.

19. Goutman JD, Calvo DJ. Studies on the mechanisms of action of picrotoxin, quercetin and pregnanolone at the GABA rho 1 receptor. Br J Pharmacol. 2004; 141:717–727. PMID: 14732759.

20. Shimada S, Cutting G, Uhl GR. gamma-Aminobutyric acid A or C receptor? gamma-Aminobutyric acid rho 1 receptor RNA induces bicuculline-, barbiturate-, and benzodiazepine-insensitive gamma-aminobutyric acid responses in Xenopus oocytes. Mol Pharmacol. 1992; 41:683–687. PMID: 1314944.

21. Johnston GA. GABA_C receptors: relatively simple transmitter-gated ion channels? Trends Pharmacol Sci. 1996; 17:319–323. PMID: 8885697.

22. Kim JH, Hong YH, Lee JH, Kim DH, Nam G, Jeong SM, Lee BH, Lee SM, Nah SY. A role for the carbohydrate portionof ginsenoside Rg3 in Na⁺ channelinhibition. Mol Cells. 2005; 19:137–142. PMID: 15750351.

23. Lee BH, Jeong SM, Lee JH, Kim DH, Kim JH, Kim JI, Shin HC, Lee SM, Nah SY. Differential effect of ginsenoside metabolites on the 5-HT_3A receptor-mediated ion current in Xenopus oocytes. Mol Cells. 2004; 17:51–56. PMID: 15055527.

24. Choi SH, Shin TJ, Hwang SH, Lee BH, Kang J, Kim HJ, Jo SH, Choe H, Nah SY. Ginsenoside Rg₃ decelerates hERG K⁺ channel deactivation through Ser631 residue interaction. Eur J Pharmacol. 2011; 663:59–67. PMID: 21586280.

25. Feigenspan A, Bormann J. Modulation of GABA_C receptors in rat retinal bipolar cells by protein kinase C. J Physiol. 1994; 481:325–330. PMID: 7738828.

26. Sine SM, Taylor P. Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells. J Biol Chem. 1982; 257:8106–8114. PMID: 7085658.

27. Lee BH, Hwang SH, Choi SH, Shin TJ, Kang J, Lee SM, Nah SY. Quercetin inhibits α3β4 nicotinic acetylcholine receptor-mediated ion currents expressed in Xenopus oocytes. Korean J Physiol Pharmacol. 2011; 15:17–22. PMID: 21461236.

28. Lee BH, Shin TJ, Hwang SH, Choi SH, Kang J, Kim HJ, Park CW, Lee SH, Nah SY. Inhibitory effects of quercetin on muscle-type of nicotinic acetylcholine receptor-mediated ion currents expressed in Xenopus oocytes. Korean J Physiol Pharmacol. 2011; 15:195–201. PMID: 21994477.

29. Shin TJ, Hwang SH, Choi SH, Lee BH, Kang J, Kim HJ, Zukin RS, Rhim H, Nah SY. Effects of protopanaxatriol-ginsenoside metabolites on rat N-methyl-d-aspartic Acid receptor-mediated ion currents. Korean J Physiol Pharmacol. 2012; 16:113–118. PMID: 22563256.

30. Wahid F, Jung H, Khan T, Hwang KH, Kim YY. Effects of red ginseng extract on visual sensitivity and ERG b-wave of bullfrog's eye. Planta Med. 2010; 76:426–443. PMID: 19830653.

31. Cheung ZH, So KF, Lu Q, Yip HK, Wu W, Shan JJ, Pang PK, Chen CF. Enhanced survival and regeneration of axotomized retinal ganglion cells by a mixture of herbal extracts. J Neurotrauma. 2002; 19:369–378. PMID: 11939504.

32. Sen S, Chen S, Wu Y, Feng B, Lui EK, Chakrabarti S. Preventive effects of North American ginseng (Panax quinquefolius) on diabetic retinopathy and cardiomyopathy. Phytother Res. 2013; 27:290–298. PMID: 22566158.