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.

Fig. 1

P2X7 antagonists impair ATP-induced ethidium⁺ uptake into RPMI 8226 cells. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the absence or presence of varying concentrations of antagonist (as indicated). Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of 1 mM ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of ethidium⁺ uptake determined by flow cytometry. Results are the mean percent of ATP-induced ethidium⁺ uptake in the absence of antagonist ± SD (triplicate data from one experiment for each antagonist)

Fig. 2

ATP-induced CD23 shedding from RPMI 8226 cells is not prevented by changes in intracellular cation concentrations. a–d Cells in NaCl medium, in a choline-Cl or b KCl medium, or in NaCl medium containing c 0.1 mM EGTA or d 50 μM BAPTA-AM were pre-incubated at 37 °C for a–c 5 min or d 30 min. a–d Cells were then incubated in the absence or presence of 1 mM ATP at 37 °C for 7 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation. Cells were labelled with PE-conjugated anti-CD23 or isotype-control mAb, and the mean fluorescence intensity (MFI) of cell-surface CD23 expression determined by flow cytometry. Results are the mean percent of ATP-induced CD23 loss ± SD (triplicate data from one experiment for each comparison); *P < 0.05 and **P < 0.01 compared to NaCl medium

Fig. 3

PLD antagonists impair ATP-induced CD23 shedding and ethidium⁺ uptake in RPMI 8226 cells. a, b Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 10 μM CAY10593, CAY10594 or halopemide. a Cells were then incubated in the absence or presence of 1 mM ATP at 37 °C for 7 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation. Cells were then labelled with PE-conjugated anti-CD23 or isotype-control mAb, and the mean fluorescence intensity (MFI) of cell-surface CD23 expression determined by flow cytometry. b Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of 1 mM ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of ethidium⁺ uptake determined by flow cytometry. Results are the a mean percent of ATP-induced CD23 loss ± SD and b mean ATP-induced ethidium⁺ uptake ± SD (triplicate data from one experiment for each antagonist); *P < 0.05 and **P < 0.01 compared to DMSO

Fig. 4

CAY10593, CAY10594 and halopemide impair ATP-induced ethidium⁺ uptake into RPMI 8226 cells in a concentration-dependent manner. Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO or varying concentrations of antagonist (as indicated). Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of 120 μM ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of ethidium⁺ uptake determined by flow cytometry. Results are the mean percent of ATP-induced ethidium⁺ uptake in the absence of compound ± SD (triplicate data from one experiment for each antagonist)

Fig. 5

CAY10593 impairs ATP-induced ethidium⁺ uptake into RPMI 8226 cells in a non-competitive-like manner. RPMI 8226 cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 2 μM or 10 μM CAY10593. Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of varying concentrations of ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of ethidium⁺ uptake determined by flow cytometry. Results are the mean percent of maximum ATP (0.5 mM)-induced ethidium⁺ uptake ± SD (data from three independent experiments)

Fig. 6

PLD1 is not required for ATP-induced ethidium⁺ uptake into RPMI 8226 cells. a RNA was isolated from A431 (positive control) and RPMI 8226 cells, and then analysed by RT-PCR using primers for PLD1 and PLD2. RNA substituted with H₂O was used as a negative control. PCR products were visualised by agarose gel electrophoresis and ethidium bromide (representative result from three independent experiments is shown). b, c RPMI 8226 cells were pre-incubated at 37 °C for 5 min in b choline-Cl or NaCl medium, or c NaCl medium in the presence of 0.27 % (v/v) 2-butanol (negative control) or 1-butanol. b, c Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of 1 mM ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and the mean fluorescence intensity (MFI) of ethidium⁺ uptake determined by flow cytometry. Results are the mean ATP-induced ethidium⁺ uptake ± SD (triplicate data from one experiment for each comparison); *P < 0.05 and **P < 0.01 compared to corresponding control

Fig. 7

CAY10593 impairs ATP-induced inward currents and pore formation in human P2X7-transfected HEK-293 cells. a Inward currents were elicited using 1 mM ATP in divalent NaCl solution. ATP was added for 5 s (denoted by black bar) before and after treatment with 10 μM CAY10593 (3–5 min). ATP was added in the continued presence of CAY10593 (single representative trace of four to seven cells is shown). b P2X7-transfected HEK-293 cells in low divalent NaCl solution containing 25 μM ethidium⁺ were pre-incubated in the presence of DMSO or CAY10593 for 15 min at 37 °C. ATP (1 mM) was injected after 40 s. Fluorescence was measured every 10 s using a plate reader (data from one experiment)

Fig. 8

CAY10593 impairs ATP-induced ethidium⁺ uptake in primary human peripheral blood mononuclear cells (PBMCs). PBMCs in NaCl medium were pre-incubated for 15 min in the presence of DMSO or 10 μM CAY10593. Cells were then incubated with 25 μM ethidium⁺ in the absence or presence of 1 mM ATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl₂ medium and centrifugation, and washed once with NaCl medium. Cells were then labelled with APC-conjugated anti-CD19. The mean fluorescence intensity (MFI) of ethidium⁺ uptake into a B cells (CD19⁺) b T cells (CD19⁻) and (c) monocytes was determined by flow cytometry. Results are the mean ATP-induced ethidium⁺ uptake ± SD (triplicate data from one donor; similar amounts of inhibition were observed in with triplicate data from a second donor, results not shown); **P < 0.01 compared to corresponding control