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BACKGROUND: Generation of the second-messenger molecule ceramide by stimulated sphingomyelinase activity has been implicated in the inflammatory processes contributing to the pathogenesis of atherosclerosis. However, reports of stimulatory effects of ceramide on endothelial NO production in animal models suggest antiatherosclerotic effects of the molecule. Therefore, we investigated long-term effects of ceramide on NO generation in human endothelial cells. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs) and in HUVEC-derived EA.hy 926 endothelial cells, C6-ceramide (N-hexanoyl-D-erythro-sphingosine) reduced the generation of bioactive NO (RFL-6 reporter-cell assay). At the same time, the signaling molecule increased endothelial NO synthase (eNOS) mRNA (RNase protection assay) and protein expression (Western blot). C6-ceramide stimulated eNOS transcription by a signaling mechanism involving protein phosphatase PP2A but did not modify the stability of the eNOS mRNA. Endothelial generation of reactive oxygen species (ROS) was increased by C6-ceramide [5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) oxidation-based fluorescence assay], and this effect was partially reversed by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). On the other hand, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) normalized in part the ceramide-induced reduction in bioactive NO. CONCLUSIONS: Ceramide produces oxidative stress in human endothelial cells, thereby reducing bioactive NO. The partial reversal of this reduction by BH(4) and the diminution of ROS generation by L-NAME suggest that ceramide promotes NADPH oxidase activity of eNOS, leading to ROS formation at the expense of NO synthesis. The ceramide-induced upregulation of eNOS gene transcription can be considered an ineffective compensatory mechanism. The decreased bioavailability of NO is likely to favor a proatherogenic role of ceramide.