Stimulation by ceramide of phospholipase A(2) 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 phos pholipase A(2) (cPLA(2)) was investigated in platelets using cell-permeable C-6-ceramide (N-hexanoylsphingosine). The addition of ceramide led to pote ntiation of thrombin-induced activation of cPLA(2) and mitogen-activated pr otein kinase (MAPK) as well as arachidonic acid release and lysophosphatidy lcholine formation. However, ceramide by itself did not induce any response . The arachidonic acid release due to the synergistic action of ceramide an d thrombin was inhibited by PD98059, a MAPK kinase inhibitor. Ceramide also stimulated thrombin-induced protein kinase C (PRC) activation, but ceramid e by itself failed to do so. Furthermore, ceramide synergistically enhanced diacylglycerol (DAG) formation and Ca2+ mobilization with thrombin, and al so DAG; formation with Ca2+-ionophore A23187, The DAG formation in response to ceramide with thrombin or A23187, as well as arachidonic acid release w ith thrombin were completely inhibited by U73122, a phospholipase C (PLC) i nhibitor. These results suggest that ceramide triggers PLC activation throu gh its synergistic action with thrombin, and subsequently potentiates the s equential PKC-MAPK cascade-cPLA(2) pathway, thus resulting in enhancement o f arachidonic acid release.