Stimulation by ceramide of phospholipase A2 activation through a mechanism related to the phospholipase C-initiated signaling pathway in rabbit platelets.
To study the involvement of sphingolipids in glycerophospholipid metabolism, the contribution of ceramide to the activation of group IV cytosolic phospholipase A2 (cPLA2) was investigated in platelets using cell-permeable C6-ceramide (N-hexanoylsphingosine). The addition of ceramide led to potentiation of thrombin-induced activation of cPLA2 and mitogen-activated protein kinase (MAPK) as well as arachidonic acid release and lysophosphatidylcholine formation. However, ceramide by itself did not induce any response. The arachidonic acid release due to the synergistic action of ceramide and thrombin was inhibited by PD98059, a MAPK kinase inhibitor. Ceramide also stimulated thrombin-induced protein kinase C (PKC) activation, but ceramide by itself failed to do so. Furthermore, ceramide synergistically enhanced diacylglycerol (DAG) formation and Ca2+ mobilization with thrombin, and also DAG formation with Ca2+-ionophore A23187. The DAG formation in response to ceramide with thrombin or A23187, as well as arachidonic acid release with thrombin were completely inhibited by U73122, a phospholipase C (PLC) inhibitor. These results suggest that ceramide triggers PLC activation through its synergistic action with thrombin, and subsequently potentiates the sequential PKC-MAPK cascade-cPLA2 pathway, thus resulting in enhancement of arachidonic acid release.