Stimulation of P2X7 receptors causes Ca2+‐ and PKC‐mediated reactive oxygen species production in murine macrophages

Abstract

Stimulation of macrophages with extracellular ATP or Bz‐ATP induces Reactive Oxygen Species (ROS) production. We tested the idea that in J774 macrophages activation of P2X7 receptors (P2X7R) by ATP leads to NADPH oxidase assembly and ROS production via Ca2+ entry and PKC activation. We used whole‐cell voltage clamp to measure P2X7R‐mediated currents, Fura‐2 to determine [Ca2+]i, 2′,7‐dichlorodihidrofluorescein to assay ROS production, PCR and western blot for P2X7R expression and phospho‐specific antibodies to determine activation of PKC isoforms. Stimulating J774 cells with Bz‐ATP (100 μM) induced an inward current at −80 mV that was sensitive to A438079 (10 μM), a blocker specific for P2X7R. ROS production induced by Bz‐ATP was also inhibited by A438079, PKC inhibitors and by removing extracellular Ca2+. Similarly, the increase in [Ca2+]i seen with Bz‐ATP was inhibited by zero external Ca2+ and A438079. Bz‐ATP induced PKC α activation. Importantly, Bz‐ATP induced a transient activation of PKC δ, while PKC ζ displayed an oscillatory pattern and was inhibited by A438079, confirming the participation of P2X7R. In summary, we propose that purinergic stimulation in macrophages induced ROS production due to Ca2+ entry via P2X7R and subsequent PKC activation which may, in turn, induce NADPH oxidase assembly and activation.Supported by grants 79897 and 105457 to JA and P. P‐C