Molecular and Cellular Pharmacology
Inhibition of ATP-induced macrophage death by emodin via antagonizing P2X7 receptor

https://doi.org/10.1016/j.ejphar.2010.04.036Get rights and content

Abstract

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an anthraquinone derivative from Rheum officinale Baill, exhibits anti-inflammatory and immunosuppressive activities, however, the underlying mechanisms are not fully understood. This study examined the effects of emodin on ATP-evoked responses in rat peritoneal macrophages and in human embryonic kidney 293 cells (HEK293) heterologously expressing the cloned rat P2X7 receptor. Emodin reduced macrophage death induced by millimolar ATP in a concentration-dependent manner with the half of maximal inhibition values (IC50) of 0.2 μM. It also strongly inhibited ATP-induced dye uptake or pore formation, a hallmark property associated with P2X7 receptor activation, and 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) induced increases in intracellular Ca2+ concentrations in macrophages with an IC50 of 0.5 μM. Furthermore, emodin significantly suppressed BzATP-evoked currents in P2X7 receptor expressing HEK293 cells with an IC50 of 3.4 μM. Taken together, these results provide compelling evidence for a novel action of emodin as a P2X7 receptor antagonist, which may underlie its anti-inflammatory and immunosuppressive activities.

Introduction

Extracellular ATP is an important signaling molecule that induces various physiological or pathological responses via activation of P2 purinergic receptors (North, 2002, Ralevic and Burnstock, 1998, White and Burnstock, 2006). Molecular and functional characterizations have divided the P2 receptors into ligand-gated ion channel P2X receptors and G-protein coupled P2Y receptors (Khakh and North, 2006, North, 2002, Ralevic and Burnstock, 1998). P2X7 receptor, which structurally belongs to the P2X receptor family, shows several hallmark functional properties (North and Surprenant, 2000, North, 2002, Surprenant et al., 1996). Millimolar concentrations of ATP are required for P2X7 activation, which is 10–100 times higher than that for other P2X and much higher than for P2Y receptors. Moreover, brief stimulation of P2X7 receptors opens Ca2+-permeable channels providing a route for extracellular Ca2+ entry into the cell. However, prolonged exposure to agonist induces “cell permeabilization” due to formation of a pore that passes large molecules including fluorescent dyes.

P2X7 receptors are highly expressed in immune cells where they mediate numerous physiological functions, such as cell proliferation and maturation (Baricordi et al., 1999, Budagian et al., 2003), cytokine release (Solle et al., 2001, Wilson et al., 2004), killing of intracellular pathogens (Fairbairn et al., 2001). Accumulating evidence also supports that activation of P2X7 mediates ATP-dependent apoptotic and/or necrotic cell death (Auger et al., 2005, Bulanova et al., 2005, Coutinho-Silva et al., 1999, Nihei et al., 2000, Taylor et al., 2008, Tsukimoto et al., 2006), which plays an important role in the immune response and inflammation. The increasing recognition of the important roles that P2X7 receptors play in immune responses, inflammation, and inflammatory pathologies has sparked enormous interest in developing potent and selective P2X7 antagonists in order to target the P2X7 receptors for therapeutic interventions (Chessell et al., 2005, Donnelly-Roberts and Jarvis, 2007, Labasi et al., 2002).

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative isolated from Rheum officinale Baill, and shows anti-inflammatory and immunosuppressive activities (Huang et al., 1991, Huang et al., 1992, Kuo et al., 2001a, Kuo et al., 2001b, Lee, 2001). The underlying mechanisms, however, are still not fully understood. Here, we have shown that emodin potently reduced ATP-induced cell death and several other P2X7 receptor-dependent responses. We have, therefore, revealed a novel action of emodin as a P2X7 receptor antagonist, which may underlie its anti-inflammatory and immunosuppressive activities.

Section snippets

Reagents

All reagents, including ATP, 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), brilliant blue G (BBG), 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) and ethidium bromide, were purchased from Sigma (St Louis, MI), except those specified individually. Emodin was from Institute of Pharmaceutical Research (Tianjin, China) or Sigma, both giving almost identical results.

Cell preparation and culture

Wistar rats (weighing 150–200 g) were sacrificed according to institutional guidelines. Hanks' balanced salt solution

Emodin inhibits ATP-induced macrophage death

We first examined ATP-induced macrophage death using the MTT assay. As shown in Fig. 1A, the percentage of dead cells induced by 0.5-1 mM ATP was slightly higher than, but not significantly different from, that in the absence of ATP. The percentage of dead cells was increased from 0 ± 2.1% under control conditions to 15.6 ± 1.1% and 36.3 ± 0.6% in the presence of 2 mM and 5 mM ATP respectively. We tested the effect of BBG, a potent rat P2X7 receptor antagonist (Jiang et al., 2000), on macrophage death

Discussion

We have shown here that emodin potently inhibits ATP-induced macrophage cell death, and P2X7 receptor-mediated responses in rat macrophages and in HEK293 cells expressing rat P2X7 receptor.

P2X7 receptors are highly and predominantly expressed in macrophages that also express other P2X and P2Y receptors (Coutinho-Silva et al., 1999, Coutinho-Silva et al., 2005). However, macrophage cell death occurred only upon exposure to high concentrations of ATP (≥ 2 mM) (Fig. 1A). Moreover, the ATP-induced

Acknowledgements

This work was supported by the Chuang Xin Fund from Nankai (to J Li) and also in part supported by a Biotechnology and Biological Science Research Council grant and Fund for International Research Collaborations from University of Leeds (to L-H Jiang). We are grateful to H Bradley for her critical comments.

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