Vanadate-induced activation of cytosolic phospholipase A2α in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na 3 VO 4 ), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of α-type cytosolic phospholipase A 2 (cPLA 2 α), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na 3 VO 4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA 2 α, were used along with a clone of C12 cells that are stably expressing cPLA 2 α (C12-cPLA 2 α cells). In the presence of a Ca 2+ ionophore (10 μM A23187), 5 and 10 mM Na 3 VO 4 synergistically stimulated AA release from L929 and C12-cPLA 2 α cells, and to a much lesser extent from control C12 cells. The release of AA by Na 3 VO 4 /A23187 was inhibited by a selective cPLA 2 α inhibitor (3 μM pyrrophenone). The release of AA by Na 3 VO 4 /A23187 was significantly inhibited by a PKC inhibitor (10 μM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 μM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 μM U0126). The phosphorylation of ERK1/2 was stimulated by Na 3 VO 4 , and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na 3 VO 4 stimulates AA release largely via cPLA 2 α activation in Ca 2+ -dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na 3 VO 4 -induced AA release from L929 cells.