CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

Abstract

The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium + uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…Furthermore, many of the original antagonists are subject to degradation when used in vivo or are predicted to have poor pharmacokinetics (Friedle et al, 2010;Jiang, 2012) , and N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide (AACBA; also known as GSK314181) (Broom et al, 2008), the cyanoguanidine derivative (Honore et al, 2006), the cyclic imide 3- [1-[[(39-nitro[1,19-biphenyl]-4-yl)oxy]methyl]-3-(4-pyridinyl) propyl]-2,4-thiazolidinedione (AZ11645373) (Stokes et al, 2006), and the nicotinamide derivative N-([4-(4-phenyl-piperazin-1-yl]tetrahydro-2H-pyran-4-yl)-methyl)-2-(phenyl-thio) nicotinamide (JNJ-47965567) (Letavic et al, 2013). The concentration responses of some of these specific antagonists (including A438079 and AZ11645373) have been determined at native murine P2X7 receptors Pupovac et al, 2013b), which is useful for their in vivo application. However, despite the availability of these newer P2X7 antagonists, many in vivo studies still use first generation antagonists (see section X).…”

Cellular and Physiological Roles for Phospholipase D1 in Cancer

“…FIPI and the isoform-selective inhibitors have become widely used to explore roles for PLD in cells (11,13,21,34,(57)(58)(59)(60)(61)(62)(63)(64)(65). To date, the inhibitors have not been associated with off-target or nonspecific effects, excepting one of the PLD1-selective compounds (66). FIPI has also been employed in vivo to study cancer growth and metastasis and platelet activation (22,35,67), which was achievable given its 5.5 h half-life and acceptable bioavailability and solubility (54).…”

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

“…Studies of P2X7-induced CD23 shedding in RPMI 8226 cells indicated that neither K + efflux, Na + influx, Ca 2+ influx nor increases in intracellular Ca 2+ were essential for this process [45]. In fact, extracellular Ca 2+ partly impaired P2X7-induced CD23 shedding from CLL cells [34].…”

“…However the mechanism by which these divalent cations impair P2X7-induced CD23 shedding remains unknown. Finally, attempts using small molecule inhibitors in RPMI 8226 cells have failed to establish a role of many enzymes, commonly downstream of P2X7 activation, in P2X7-induced CD23 shedding including PKC, JNK, Rho kinase, PI3K, GSK-3, MAPK, acid sphingomyelinase and phospholipases [45].…”