Activation Of Acid Sphingomyelinase Drives Lysosomal Trifficking And Fusion To Membrane In Coronary Endothelial Cells

Recent studies have demonstrated that activation of death receptor results in lysosomal fusion to cell membrane to form a lipid rafts (LRs) signaling platform. However, the driving forces are still unknown. The present study was designed to test a hypothesis that acid sphingomyelinase (ASMase) is firstly activated to produce ceramide, then drives lysosomal fusion and LRs clustering. We first performed confocal microscopy and fluorescence resonance energy transfer (FRET) detection in bovine coronary arterial endothelial cells (BCAECs) and found that FasL significantly led to colocalization of Lamp‐1 and CTX‐B, and their FRET efficiency increased from 5 to 19%. We also found a lysosome‐membrane fusion pattern of fluorescence changes induced by FasL which was blocked by siRNA of ASMase. Both activator and inducer of ASMase markedly increased colocalization of Lamp‐1 with CTX‐B and resulted in lysosome‐membrane fusion. However, the de novo synthesis of ceramide had no such effects. In isolated bovine coronary arteries, endothelium‐dependent vasodilation was impaired after ASMase had been activated, which could be reversed by lysosome functional inhibitor. It is concluded that ASMase activation precedes lysosomal fusion and LRs clustering and may serve as a driving force for lysosome trafficking and fusion to cell membrane in BCAECs. (Supported by NIH Grants HL‐57244, HL‐75316, and DK54927).