Potentiation of Native and Recombinant P2X7-Mediated Calcium Signaling by Arachidonic Acid in Cultured Cortical Astrocytes and Human Embryonic Kidney 293 Cells

Abstract

In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of [Ca(2+)](i) in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced [Ca(2+)](i) elevation is coupled to depletion of intracellular Ca(2+) stores and to a sustained noncapacitative Ca(2+) entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca(2+)](i) rise evoked by the selective agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked [Ca(2+)](i) elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in [Ca(2+)](i) was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient [Ca(2+)](i) elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced [Ca(2+)](i) rise. Together, these findings indicate that AA mediates a complex regulation of [Ca(2+)](i) dynamics also through P2X7-mediated Ca(2+) entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca(2+)](i) signaling in astroglial cells.