Human placenta sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase, induces oxidative stress, glutathione depletion, and apoptosis in rat hepatocytes.
Ceramide has been identified as a putative lipid messenger that mediates diverse cellular processes including cell death. Since glutathione (GSH) depletion is known to sensitize cells to many cytotoxic agents and as a result of the reported regulation of neutral sphyngomyelinase (NSMase) by GSH, the present study compared the role of individual SMases in the induction of oxidative stress, regulation of cellular GSH, and apoptosis of rat hepatocytes. Exposure of cultured rat hepatocytes to exogenous Bacillus cereus sphingomyelinase (bSMase), a neutral SMase, or human placenta sphingomyelinase (hSMase), an acidic SMase (ASMase), generated similar ceramide levels in a dose-dependent manner. However, whereas bSMase increased hepatocellular GSH levels, hSMase depleted GSH stores, an effect that was prevented by monensin and mannose 6-phosphate (M-6-P), suggesting that exogenous hSMase enters hepatocytes by endocytosis and is delivered to an endosomal/lysosomal acidic compartment. Interestingly, despite the differential effect of either SMases on cell GSH levels, both bSMase and hSMase increased gamma-glutamylcysteine synthetase heavy-subunit chain (gamma-GCS-HS) mRNA levels. Consistent with these findings on GSH regulation, hSMase, but not bSMase, generated reactive oxygen species (ROS), being accompanied by mitochondrial depolarization, suggesting that hSMase targeted mitochondria, leading to oxidative stress. Accordingly, hepatocytes displayed a selective sensitivity to hSMase in contrast to bSMase exposure, and depletion of GSH stores enhanced susceptibility to hSMase as a result of potentiation of ROS formation and caspase 3 activation. Thus, these findings reveal the ability of ASMase to induce oxidative stress as a result of the targeting of mitochondria, and that GSH depletion sensitizes hepatocytes to the ASMase-induced apoptosis.
García-Ruiz
C
C
Liver Unit, Instituto Malalties Digestives, Hospital Clinic i Provencial, Instituto de Investigaciones Biom¿edicas, August Pi i Sunyer, Consejo Superior Investigaciones Cient¿ificas, Barcelona 08036, Barcelona, Spain.
Marí
M
M
Morales
A
A
Colell
A
A
Ardite
E
E
Fernández-Checa
J C
JC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002518:Ceramides / Q000032:analysis; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D010920:Placenta / Q000201:enzymology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*
D002518:Ceramides; D017382:Reactive Oxygen Species; D013108:Sphingomyelin Phosphodiesterase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D008274:Magnesium