Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thu s may contribute to acute lytic phases as well as to resolution of glomerul onephritis. However, the mechanism involved in these events is still unclea r. We report here that chronic exposure of renal mesangial cells for 24 h t o compounds releasing NO, including spermine-NO, (Z)-1-(N-methyl-N-[6-(N-me thylammoniohexyl)-amino])diazen-1-ium-1,2-diolate (MAHMA-NO), S-nitrosoglut athione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceram ide, Time courses reveal that significant effects occur after 2-4 h of stim ulation with NO donors and reach maximal levels after 24 h of stimulation, No acute (within minutes) ceramide production can be detected. When cells w ere stimulated with NO donors in the presence of phorbol ester, a direct ac tivator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide part ially reversed the inhibitory effect of phorbol ester on apoptosis, thus su ggesting a negative regulation of protein kinase C on ceramide formation an d apoptosis, In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within m inutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNF alpha cause a chronic activation of acidic and ne utral sphingomyelinases, the ceramide-generating enzymes, whereas acidic an d neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNF alpha, Furthermore, in the presence of a n acidic ceramidase inhibitor, N-oleoylethanolamine, TNF alpha leads to a s ustained accumulation of ceramide and in parallel induces DNA fragmentation . In summary, our data demonstrate that exogenous NO causes a chronic up-regu lation of ceramide levels in mesangial cells by activating sphingomyelinase s and concomitantly inhibiting ceramidases, and that particularly the late- phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.