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Nitric oxide (NO) in apoptotic versus necrotic RAW 264.7 macrophage cell death: the role of NO-donor exposure, NAD+ content, and p53 accumulation.

Nitric oxide (NO)-releasing compounds cause apoptotic cell death in RAW 264.7 macrophages. This is confirmed morphologically by chromatin condensation and biochemically by DNA laddering. With use of spontaneously decomposing NO donors known as NONOates we show that the integral of concentration over time accounts for the NO-donor damaging ability. A 30-min exposure to the rapidly decomposing NO-donor diethylamine-nitric oxide complex (DEA-NO) causes irreversible damage and apoptotic cell death after 6 to 8 h. For intermediate NO releasers like sodium nitroprusside, S-nitrosoglutathione (GSNO), and spermine-NO removal of the NO-donating compound halts fragmentation to a certain degree. The relatively stable diethylenetriamine-nitric oxide complex initiates fragmentation only after prolonged exposure. NO-mediated apoptotic signaling in macrophages neither decreases cellular NAD+, nor causes a drop in APT. Consistently, membrane integrity measured by lactate dehydrogenase release is preserved and inhibitors of poly(ADPribose) polymerase, like 3-aminobenzamide, are noneffective. The level of the tumor suppressor p53 increases in response to NO donors like GSNO and effectively senses NO intoxication in macrophages. GSNO removal concomitantly allows p53 to decline with only a small percentage of cells showing DNA fragmentation. Contrary, massive damage initiated by a 1-h exposure to DEA-NO is irreversible, with persistent p53 levels. NO-mediated apoptotic cell death in RAW 264.7 macrophages correlates with the degree of p53 accumulation, probably sensing the integrity of the genome.