Premium
ATP‐induced arachidonic acid release in cultured astrocytes is mediated by Gi protein coupled P2Y 1 and P2Y 2 receptors
Author(s) -
Chen Wei C.,
Chen ChingC.
Publication year - 1998
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/(sici)1098-1136(199804)22:4<360::aid-glia5>3.0.co;2-7
Subject(s) - biology , pertussis toxin , arachidonic acid , purinergic receptor , p2y receptor , ppads , receptor , adenosine , protein kinase c , adenosine triphosphate , activator (genetics) , medicine , endocrinology , phosphorylation , biochemistry , g protein , enzyme
ATP‐induced arachidonic acid (AA) release was studied in [ 3 H]AA‐prelabeled cultured astrocytes. To characterize the P 2 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 [ 3 H]AA release, with EC 50 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 P2Y 1 and P2Y 2 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. Ca 2+ 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 [Ca 2+ ] i and a sustained [Ca 2+ ] i increase. The AA release was blocked in the external Ca 2+ free in condition the sustained [Ca 2+ ] i increase was abolished. Both A23187‐ and TPA‐induced AA release were also blocked in this condition. Furthermore, inorganic Ca 2+ channel blocker Co 2+ 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 A 2 (cPLA 2 ). 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 cPLA 2 induced by A23187 were reversed in the absence of external Ca 2+ , suggesting the involvement of PKCα in MAPK activation and mobility shift of cPLA 2 . Taken together, ATP‐stimulated AA release was secondary to the activation of P2Y 1 and P2Y 2 receptors/PLC pathway. Ca 2+ and PKC interact to regulate this response. Elevation of intracellular Ca 2+ , the mechanism involving extracellular Ca 2+ influx, might act partly through PKCα activation and in turn MAPK might be activated, leading to cPLA 2 phosphorylation and AA release. GLIA 22:360–370, 1998. © 1998 Wiley‐Liss, Inc.