Received for publication October 21, 2005.
Revised February 9, 2006.
Accepted for publication March 1, 2006.

POTENTIATION OF NATIVE AND RECOMBINANT P2X7-MEDIATED CALCIUM SIGNALING BY ARACHIDONIC ACID IN CULTURED CORTICAL ASTROCYTES AND HEK293 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 microfluorimetry we investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of free intracellular calcium ([Ca²⁺]_i) in cultured neocortical type-1 astrocytes and P2X7, P2X2 transfected HEK293 cells. We report that in cultured astrocytes, AA-induced [Ca²⁺]_i elevation is coupled to depletion of intracellular Ca²⁺ stores and to a sustained non-capacitative Ca²⁺ entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca²⁺]_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 [Ca²⁺]_i elevation. Fluorescent dye uptake experiments showed that the AA- induced increase in [Ca²⁺]_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 rat P2X2. Control HEK293 cells responded to AA exposure only with a transient [Ca²⁺]_i elevation whereas in those expressing P2X7 receptors, AA elicited a potentiation of the BzATP-induced [Ca²⁺] _i rise. Altogether, these findings indicate that AA mediates a complex regulation of [Ca²⁺]_i dynamics also through modulation of P2X7-mediated Ca²⁺ entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca²⁺]_i signalling in astroglial cells.

Key words: Purinergic, Ca imaging, Regulation - physiological, Eicosanoids, Ischemia/Reperfusion

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