Nitric oxide stimulates chronic ceramide formation in glomerular endothelial cells

Exposure of glomerular endothelial cells for 24 h to compounds releasing NO , including spermine-NO, MAHMA-NO, and S-nitroso-glutathione, results in a dose-dependent and delayed (after 24 h) increase in the lipid signaling mol ecule ceramide. This NO-induced stimulation occurs in a cGMP-independent fa shion since the membrane-permeant cGMP analogue dibutyryl cGMP has no effec t on chronic cer amide production. Short-term incubation of endothelial cel ls for 20 min reveals that NO and dibutyryl cGMP fail to stimulate an acute ceramide increase, whereas TNF-alpha, a well-known activator of sphingomye linases, is able to acutely increase ceramide formation. Interestingly, N-o leoylethanolamine, an acidic ceramidase inhibitor, potentiates NO-induced c hronic ceramide production, indicating that ceramide generation rather than ceramide metabolism is modulated by NO. Furthermore, NO-induced delayed ce ramide formation is partially inhibited by the thiol-specific inhibitor iod oacetamide and the radical scavenger alpha-tocopherol, suggesting a regulat ory role of thiol-containing enzymes and the involvement of a redox-sensiti ve mechanism. In addition, NO causes an increased DNA fragmentation in glom erular endothelial cells which is further enhanced by N-oleoylethanolamine and can be mimicked by exogenous ceramide. In summary, these results imply that ceramide represents an important mediator of NO-triggered chronic cell responses like apoptosis. Inhibition of ceramide synthesis may provide a n ew therapeutic approach to the treatment of pathological conditions involvi ng increased NO formation. (C) 1999 Academic Press.