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J Biol Chem, Vol. 273, Issue 12, 6921-6927, March 20, 1998
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From the Departments of PC12 cells undergo apoptosis as well as necrosis
following exposure to hypoxia. Following a 6-h hypoxic treatment, a
time-dependent increase in intracellular ceramide level was
observed with a concurrent decrease in sphingomyelin. It was also shown
that the hypoxia-induced ceramide accumulation resulted from activation
of neutral magnesium-dependent sphingomyelinase.
Comparative kinetic analyses of the neutral sphingomyelinase in the
cells under normoxia and hypoxia showed that hypoxia increased
Vmax but did not affect Km
of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and
activation of neutral sphingomyelinase was reduced. Addition of
exogenous C2-ceramide induced cell death and activated
caspase-3 as markedly as the hypoxia treatment. On the other hand, in
PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3
prevented cell death by either hypoxia or C2-ceramide.
These results suggest that ceramide generated by activation of neutral
magnesium-dependent sphingomyelinase mediates hypoxic cell
death and that Bcl-2 has inhibitory effects on ceramide formation and
caspase activation.
Neurosurgery and
¶ Biochemistry, Gifu University School of Medicine,
Tsukasamachi-40, Gifu 500-8705, Japan and the Department of
Medical Genetics, Biomedical Research Center, Osaka University
Medical School, Suita 565, Japan
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