Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors.
OBJECTIVE
The P2X7 receptor is an ATP-gated ion channel predominantly expressed in immune cells and plays a key role in inflammatory processes. Ginseng is a well-known Chinese herb with both pro- and anti-inflammatory properties and many of its actions have been ascribed to constituent ginsenosides. We screened a number of ginsenoside compounds for pharmacological activity at P2X7 receptors, that might contribute to the reported immunomodulatory actions of ginseng.


METHODS
We used several assays to measure responses of P2X7 receptors, ATP-mediated dye uptake, intracellular calcium measurement and whole-cell patch-clamp recordings. HEK-293 cells stably expressing human P2X7 receptors were used in addition to mouse macrophages endogenously expressing P2X7 receptors.


RESULTS
Four ginsenosides of the protopanaxdiol series, Rb1, Rh2, Rd and the metabolite compound K (CK) potentiated the dye uptake responses of P2X7 receptors, whereas other ginsenosides tested were ineffective (1-10 μM). The potentiation was rapid in onset, required a threshold concentration of ATP (>50 μM) and had an EC50 of 1.08 μM. CK markedly enhanced ATP-activated P2X7 currents, probably via an extracellular site of action. One of the consequences of this potentiation effect is a sustained rise in intracellular Ca(2+) that could account for the decrease in cell viability in mouse macrophages after a combination of 500 μM ATP and 10 μM CK that are non-toxic when applied alone.


CONCLUSIONS
This study identifies selected ginsenosides as novel potent allosteric modulators of P2X7 channels that may account for some of the reported immune modulatory actions of protopanaxdiol ginsenosides in vivo.
School of Health Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
R M
RM
Helliwell
School of Medical Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
C O
CO
ShioukHuey
School of Medical Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
K
K
Dhuna
School of Health Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
J C
JC
Molero
School of Health Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
J-M
JM
Ye
School of Health Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.
C C
CC
Xue
School of Medical Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Victoria, Australia.|School of Pharmacy, University of East Anglia, Norwich, UK.
L
http://orcid.org/0000-0003-4013-6781
L
Stokes
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001583:Benzoxazoles / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D036145:Ginsenosides / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D011806:Quinolinium Compounds / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D012502:Sapogenins
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D036145:Ginsenosides; D011806:Quinolinium Compounds; D058486:Receptors, Purinergic P2X7; D012502:Sapogenins; C089813:YO-PRO 1; D000255:Adenosine Triphosphate; C062916:protopanaxadiol; D002118:Calcium