ATP-induced arachidonic acid release in cultured astrocytes is mediated by Gi protein coupled P2Y1 and P2Y2 receptors

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Abstract
ATP‐induced arachidonic acid (AA) release was studied in [3H]AA‐prelabeled cultured astrocytes. To characterize the P2 purinoceptor‐mediated effect of ATP, the subtype‐specific agonists 2‐methylthio ATP (2‐MeSATP) and UTP were compared. ATP, UTP, or 2‐MeSATP induced a dose‐dependent increase of [3H]AA release, with EC50 values of 22.7 μM, 29.4 μM, and 1.68 μM, respectively; α,β‐methylene ATP and adenosine had no effect. The order of potency was ATP = UTP ≥ 2‐MeSATP, indicating that ATP interacted with both P2Y1 and P2Y2 receptors to mediate AA release in astrocytes. The effect of ATP, UTP, or 2‐MeSATP was markedly inhibited by pretreatment of cells with pertussis toxin. Ca2+ ionophore‐A23187 and PKC activator‐TPA mimicked the effects of these three agonists to stimulate AA release. ATP, UTP, and 2‐MeSATP induced a rapidly initial rise of [Ca2+]i and a sustained [Ca2+]i increase. The AA release was blocked in the external Ca2+ free in condition the sustained [Ca2+]i increase was abolished. Both A23187‐ and TPA‐induced AA release were also blocked in this condition. Furthermore, inorganic Ca2+ channel blocker Co2+ inhibited ATP, UTP, or 2‐MeSATP induced AA release as well. Long‐term (24 h) treatment of cells with TPA resulted in an attenuation of three agonists, TPA or A23187 response. Similarly, ATP or TPA promoted AA release was inhibited by the mitogen‐activated protein kinase (MAPK) cascade inhibitor PD 98059. ATP, TPA, or A23187 induced an increase in the activity and tyrosine phosphorylation of p42 MAPK, as well as a molecular weight shift, consistent with phosphorylation, of cytosolic phospholipase A2 (cPLA2). ATP‐ and TPA‐stimulated activation of p42 MAPK activity and tyrosine phosphorylation were inhibited by long‐term TPA treatment, while A23187‐stimulated effects were completely blocked. Furthermore, tyrosine phosphorylation and activation of p42 MAPK and mobility shift of cPLA2 induced by A23187 were reversed in the absence of external Ca2+, suggesting the involvement of PKCα in MAPK activation and mobility shift of cPLA2. Taken together, ATP‐stimulated AA release was secondary to the activation of P2Y1 and P2Y2 receptors/PLC pathway. Ca2+ and PKC interact to regulate this response. Elevation of intracellular Ca2+, the mechanism involving extracellular Ca2+ influx, might act partly through PKCα activation and in turn MAPK might be activated, leading to cPLA2 phosphorylation and AA release. GLIA 22:360–370, 1998.