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 RA W 264.7 macrophages. This is confirmed morphologically by chromatin co ndensation and biochemically by DNA laddering, With the use of spontan eously decomposing NO donors known as NONOates we show that the integr al of concentration over time accounts for the NO-donor damaging abili ty, A 30-min exposure to the rapidly decomposing NO-donor diethylamine -nitric oxide complex (DEA-NO) causes irreversible damage and apoptoti c cell death after 6 to 8 h. For intermediate NO releasers like sodium nitroprusside, S-nitrosoglutathione (GSNO), and spermine-NO removal o f the NO-donating compound halts fragmentation to a certain degree, Th e relatively stable diethylenetriamine-nitric oxide complex initiates fragmentation only after prolonged exposure. NO-mediated apoptotic sig naling in macrophages neither decreases cellular NAD(+), nor causes a drop in ATP, Consistently, membrane integrity measured by lactate dehy drogenase release is preserved and inhibitors of poly(ADPribose) polym erase, like 3-aminobenzamide, are noneffective. The level of the tumor suppressor p53 increases in response to NO donors like GSNO and effec tively senses NO intoxication in macrophages. GSNO removal concomitant ly allows p53 to decline with only a small percentage of cells showing DNA fragmentation. Contrary, massive damage initiated by a l-h exposu re to DEA-NO is irreversible, with persistent p53 levels, NO-mediated apoptotic cell death in RAW 264.7 macrophages correlates with the degr ee of p53 accumulation, probably sensing the integrity of the genome. (C) 1996 Academic Press, Inc.