We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor- (TNF-) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30% inhibition) when challenged with TNF- or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-, its levels were measured at various times after the TNF- preconditioning. We present evidence for the first time that, in normal brain cells, TNF- pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-. The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-: 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B₁, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.

ceramide; intercellular adhesion molecule-1; astrocytes; brain endothelial cells; ischemic tolerance; tumor necrosis factor-

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