Controversy exists regarding the nature of the "executioner" sphingomyelinase (SMase) in cells and its subcellular localization. A new fluorescence-based assay with the substrate 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine allowed rapid and reliable microassays of neutral (N) and acid (A) SMase activity in cell extracts from primary cultures of neonatal rat oligodendrocytes (OPC) and a human oligodendroglioma cell line (HOG). Total SMase activity was much higher in OPC than in HOG cells. Both staurosporine and tumor necrosis factor-alpha (TNF-alpha) induced apoptosis and activated NSMase in a multiphasic manner in both OPC and HOG cells. The increase in caspase 8 activity preceded the 1 hr peak of NSMase activation, which was followed by caspase 3 activation. In contrast, ASMase activity, which constituted >90% of the total SMase activity, was unresponsive to proapoptotic drugs. Neither reducing ASMase levels by 50% by pretreatment with desipramine nor inhibiting sphingolipid synthesis by 50% with fumonisin B1 had any effect on cell death. Isolation of sphingolipid-rich plasma membrane microdomains (rafts) from the cells by sucrose density gradient ultracentrifugation revealed an enrichment of sphingomyelin, ceramide, and caspase 8. Proapoptotic drugs such as staurosporine promoted the translocation of NSMase to the raft fraction. In contrast, ASMase, other lysosomal hydrolases, and caspase 3 remained absent from rafts even after staurosporine treatment. The staurosporine-induced concomitant increase of ceramide in the raft fraction and caspase 3 in the cytosol could be mimicked by the addition of exogenous bacterial SMase. We conclude that caspase 8 activates NSMase in rafts in oligodendrocytes and that the downstream apoptotic signal is via caspase 3.

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [ 14 C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P 3 ] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P 2 ]. When PtdIns(3,4,5)P 3 was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P 3 to PtdIns(4,5)P 2 , we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P 3 levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P 3 and is most likely involved in receptor clustering and capping. Keywords: phosphate, phosphatidylinositol-3-kinase, phosphatidyl-myo-inositol 3,4,5-triphosphate, PTEN, sphingomyelinase. Abbreviations used: ASMase, acid sphingomyelinase; DISC, deathinducing signaling complex; HMU-P-Chol, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine; HOG, human oligodendroglioma cells; Ins(1,4,5)P 3 , myo-inositol 1,4,5-triphosphate; lyso-PA, lysophosphatidic acid; NGF, nerve growth factor; NSMase, neutral sphingomyelinase; NT-3, neurotropin-3; NTR, neurotropin receptor; OPC, oligodendrocyte primary cultures; PI3 kinase, phosphatidylinositol-3-kinase; PtdIns(4,5)P 2 , phosphatidyl-myo-inositol 4,5-diphosphate; PtdIns(3,4,5)P 3 , phosphatidyl-myo-inositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog protein; Raft, Triton X-100-insoluble, sphingolipid-rich membrane fraction from sucrose density gradient; TNF, tumor necrosis factor.