Abstract
Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.
Highlights
nitric oxide (NO) and tumor necrosis factor (TNF)␣ cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNF␣
Renal mesangial cells exposed to proinflammatory cytokines like interleukin-1 or tumor necrosis factor-␣ express an inducible nitric-oxide synthase (NOS) and produce large amounts of NO [5, 6], which may contribute to certain forms of glomerulonephritis [4, 7, 8]
Spots corresponding to ceramide and sphingosine were analyzed and quantitated using a Berthold (Nashua, NH) LB 2842 automatic TLC scanner. It is well documented from our previous studies that glomerular mesangial cells and endothelial cells are able to undergo apoptosis upon stimulation with exogenous NO donors [9, 10]
Summary
NO and TNF␣ cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNF␣. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished.
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