NATIVE AND RECOMBINANT P2X7-MEDIATED CALCIUM SIGNALLING: SPECIFIC MODULATION BY ARACHIDONIC ACID

S. Alloisio1, S. Ferroni2 and M. Nobile1
1Institute of Biophysics, CNR, Via De Marini, 6 – 16149 Genoa, Italy
 alloisio@ge.ibf.cnr.it
2Dept. of Human and General Physiology, University of Bologna, Via San Donato, 19/2 – 40127 Bologna, Italy

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 no rat P2X2. Moreover, in HEK293 AA did not induce potentiation of P2X7–mediated [Ca2+]i rise when the cytosolic C-terminal region of P2X7 receptor was truncated. Altogether, these findings indicate that AA mediates a complex regulation of [Ca2+]i dynamics also through P2X7-mediated Ca2+ entries, 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.  Supported by MIUR (Italy)