Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor‐expressing cells: implications for its therapeutic action

Abstract

Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTSDisrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1b release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1b, interferon-g and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONSColchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action -inhibition of pore formation induced by activation of P2X receptors -which could explain some of the anti-inflammatory effects of colchicine. LINKED ARTICLEThis article is commented on by Pelegrín, pp. 908-911 of this issue. To view this commentary visit http://dx.doi.org/10.1111/ j. 1476-5381.2011.01325.x Abbreviations DAPI, 4′,6-diamidino-2-phenylindole; DCFH2DA, dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; EB, ethidium bromide; IFN-g, interferon-g; IL-1b, interleukin-1b; LPS, lipopolysaccharide; PGP, P-glycoprotein; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species BJP British Journal of Pharmacology

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

“…CAY10593 was originally synthesised via the modification of the 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one analogue halopemide, but unlike halopemide, CAY10593 contains a chiral (S)-methyl group which prompts PLD1 preferring pharmacology [30]. CAY10594, which has a 1-phenyl-1,3,8-triazaspiro [4,5]decan-4-one scaffold instead of a 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one scaffold, also lacks a chiral (S)-methyl group [30]. Therefore, this structural group may be of importance in the interaction of CAY10593 with P2X7.…”

“…Prolonged exposure of P2X7 to extracellular ATP opens a second permeability state or pore that allows the uptake of organic cations including fluorescent dyes such as ethidium + [2]. Whether this second permeability state is attributed to intrinsic channel dilation [3], the pannexin-1 channel [4] or an alternate but unknown uptake pathway [5][6][7] remains controversial. Moreover, our understanding of this permeability state is further complicated with some [8][9][10] but not other [11][12][13] studies showing that P2X7-induced dye uptake involves the p38 mitogen-activated protein kinase.…”

The Therapeutic Potential of Modifying Inflammasomes and NOD-Like Receptors

“…The pathogenetic role of dysregulated caspase-1 in FMF is demonstrated by the clinical efficacy of IL-1b inhibition in patients refractory to colchicine, which remains the drug of choice (Stankovic Stojanovic et al, 2012;Ter et al, 2012). Interestingly, one of the as-yet-illcharacterized effects of colchicine in immune cells is to block P2X7R and P2X7R-associated IL-1b release (Marques-da- Silva et al, 2011), an effect that might be relevant to the anti-inflammatory activity of this drug.…”

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…In addition, threonine 283 (Thr283) has been described as a critical residue in the ectodomain for P2X7R receptor function and it has been suggested that the intracellular leucine residue (P451L) alters downstream signalling independently of ion channel activity (Young et al, 2006). Recently, Marques-da-Silva and collaborators (Marques-da-Silva et al, 2011) demonstrated that colchicine did not inhibit ATP-evoked currents in macrophages, but it decreased ATP-induced dye uptake. Large conductance channel opening on Xenopus oocytes and HEK293 cells expressing P2X7R were inhibited after colchicines treatment (Marques-da-Silva et al, 2011).…”

“…This information is based on the partial blockage of the P2X7R receptor induced dye uptake exhibited after pannexin-1 inhibition Locovei et al, 2007;Pelegrin & Surprenant, 2007). Alternatively, other large conductance channel such as MTX-induced pore (Pelegrin & Surprenant, 2007), P2X2R large conductance channel (Marques-da-Silva et al, 2011) and the rising of intracellular Ca 2+ induced pore (Faria et al, 2009) were not impaired by pannexin-1 inhibitors.…”