Regulation of phosphatidylserine exposure at the cell surface by the serine-base exchange enzyme system during CD95-induced apoptosis

Early in the apoptotic process, CD95 induces a translocation of phosphatidy lserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium -dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis o n CD95-induced PtdSer exposure at the surface of Jurkat cells. We found tha t CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompan ied both by a decreased PtdSer synthesis and by a strong reduction of CD95- induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V-fluorescein isothiocyanate (FITC) -labeled cells. PtdSer synthesis through the serine-base exchange enzyme sy stem thus appeared as one of the mechanisms implicated in the recently disc overed CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversel y, increasing the activity of the serine-base exchange enzyme system with d ifferent drugs, either the K+ channel blocker quinine, the cationic amphiph il stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide ( W7), resulted in an increased appearance of PtdSer at the surface of CD95-t reated cells. Both PtdSer synthesis and CD95-induced annexin V-FITC reactiv ity were abrogated in ATP depleted cells. Also, modifying the membrane pote ntial with valinomycin (hyperpolarization) or either gramicidin or KCL (dep olarization) demonstrated a strong relationship between PtdSer synthesis an d annexin V-FITC reactivity in CD95-treated cells. Together, our results in dicate that CD95-induced exposure of PtdSer at the cell surface could be re gulated by the activity of the serine-base exchange enzyme system. BIOCHEM PHARMACOL 59;7:855-863, 2000. (C) 2000 Elsevier Science Inc.