Release of arachidonic acid by 2-arachidonoyl glycerol and HU210 in PC12 cells; roles of Src, phospholipase C and cytosolic phospholipase A2α

The phospholipase A2 (PLA2)-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (α type cytosolic PLA2 (cPLA2α), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA2α, the activities of Src family kinases, Ca2+-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 μM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB1 receptor) and by pyrrophenone (a selective inhibitor of cPLA2α) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA2α and an increase in the intracellular Ca2+ level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca2+ response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA2α in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB1 receptor and/or CB1-like receptor in neuronal cells.