Ceramide induces a loss in cytosolic peroxide levels in mononuclear cells.

Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313-11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the peroxide (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in peroxide, as measured by the peroxide-sensitive probe 2',7'-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in peroxide was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N -acetyl cysteine or glutathione conferred apoptosis. However, N -acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the peroxide of both primary and immortalized cells, the magnitude of which dictates the cellular response.