Hydrogen peroxide-induced arachidonic acid release in L929 cells; roles of Src, protein kinase C and cytosolic phospholipase A2alpha.
Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.
Taniguchi
Tomoko
T
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chiba 260-8675, Japan.
Shimizu
Masaya
M
Nakamura
Hiroyuki
H
Hirabayashi
Tetsuya
T
Fujino
Hiromichi
H
Murayama
Toshihiko
T
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D054513:Group IV Phospholipases A2; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C412373:AG 1879; D004791:Enzyme Inhibitors; D007211:Indoles; D007476:Ionophores; D008301:Maleimides; D011743:Pyrimidines; D011759:Pyrrolidines; C455981:pyrrophenone; D016718:Arachidonic Acid; D000001:Calcimycin; D006861:Hydrogen Peroxide; C087857:protein kinase C gamma; D019061:src-Family Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium