Journal of Cell Science 110, 2315-2322 (1997) 2315 Printed in Great Britain © The Company of Biologists Limited 1997 JCS4287 Differential induction of apoptosis in Swiss 3T3 cells by nitric oxide and the nitrosonium cation Shazia Khan1,2, Midori Kayahara1,2, Umesh Joashi2, Nicholas D. Mazarakis2, Catherine Sarraf3, A. David Edwards2, Martin N. Hughes1 and Huseyin Mehmet2,* 1Department of Chemistry, King’s College London, Strand, London WC2R 2LS, UK 2Weston Laboratory, Department of Paediatrics and Neonatal Medicine, and 3Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK *Author for correspondence (e-mail: [email protected]) SUMMARY We have investigated the effect of nitric oxide (NO) on decomposition to NO. The apoptotic effect of NO was apoptosis in Swiss 3T3 fibroblasts and compared it to the reduced in the presence of the NO scavenger oxyhaemo- effect of the nitrosonium cation (NO+). Both species globin, or the antioxidants N-acetylcysteine and ascorbic induced apoptosis, confirmed by electron microscopy, acid, whereas in the case of NO+ these antioxidants poten- propidium iodide staining, DNA laddering and activation tiated apoptosis. Glutathione also had a potentiating effect of caspases. The kinetics of triggering apoptosis were on the cytotoxicity of NO+. This suggests that cellular different for the two redox species: NO+ required only a 2 antioxidants may play a role in protecting the cell from NO- hour exposure, whereas NO required 24 hours. Three induced apoptosis while NO+ may trigger apoptosis inde- sources of NO were used: aqueous solutions of NO and two pendently of oxidative stress mechanisms. NO donors, S-nitrosoglutathione and S-nitroso-N- acetylpenicillamine. The time course of apoptosis induced by these two S-nitrosothiols correlated with their rate of Key words: Apoptosis, Nitric oxide, Mitochondrion INTRODUCTION NO to be readily oxidised or reduced to species with chemi- cally distinct properties and potentially different biological Nitric oxide (nitrogen monoxide, NO)1 is a key molecule in effects. For example, in the nervous system, NO can be neu- intracellular signal transduction. Its ability to activate rotoxic, in part through its reaction with the superoxide ion − − guanylate cyclase is well recognised but NO may also regulate (O2 ) to form peroxynitrite (ONO2 ). This is a highly reactive other signalling pathways; for example, NO can modify molecule that can induce lipid peroxidation or inactivate iron- enzyme function by nitrosating thiol groups in cellular sulphur clusters (Radi et al., 1991; Hausladen and Fridovich, enzymes (Ignarro, 1992; Stamler, 1994) and can itself act as a 1994). In contrast, the nitrosonium cation (NO+) can act as a neurotransmitter in both the central and peripheral nervous protective signal (Lipton et al., 1993), suggesting that the redox systems (Snyder, 1992). Since NO can also act as a cytotoxic state of NO influences NO-mediated effects. agent in the immune and nervous systems (Hibbs et al., 1987; NO+ is produced by the one-electron oxidation of NO. Dawson et al., 1991) this property, together with its role in Although it is an effective nitrosating agent at low pH, at phys- intracellular signalling, is consistent with NO inducing cell iological pH NO+ is a transient species with an estimated half- death by apoptosis. life of 10−10 seconds, being immediately hydrolysed to nitrite. Apoptotic cell death is an active physiological process However, carriers of the NO+ group may exist within the cell; distinct from necrosis and is characterised by defined morpho- these include nitrosylhaems and S-nitrosothiols (Wade and logical and biochemical features including membrane Castro, 1990; Barnett et al., 1995). blebbing, cytoplasmic shrinkage, nuclear pyknosis and DNA The aim of this study was to investigate the toxicity of NO fragmentation (Kerr et al., 1972; Martin et al., 1994). However, and NO+ on Swiss 3T3 fibroblasts. These cells are well char- the signalling pathways that lead to apoptosis remain poorly acterised at the intracellular signalling level and can be understood. NO might induce apoptosis through its interactions reversibly arrested in the G0 phase of the cell cycle, thus with membrane or cytosolic targets, or act as an intracellular removing the heterogeneity of response that may exist in an messenger in the apoptotic cascade. However, recent reports asynchronous population (Rozengurt, 1986). Aqueous have shown that, while NO can induce apoptosis in activated solutions of NO, or the NO-donors S-nitrosoglutathione macrophages, it prevents apoptosis in other cell lines (Cui et (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were al., 1993; Mannick et al., 1994; Farinelli et al., 1996). used. The effect of NO+ was investigated using the nitroprus- 2− These conflicting data could be explained by the ability of side anion ([Fe(CN)5(NO)] ). This molecule contains a co- 2316 S. Khan and others ordinated NO+ ligand and acts as a donor of NO+ at physio- All experiments with SNP were performed in the dark to prevent logical pH (Bates et al., 1991). NO or NO+-induced apoptosis photolysis of the nitroprusside anion to produce NO. Cycloheximide was also investigated in the presence of the NO-scavenger oxy- (CHX, 5 µg/ml), a protein synthesis inhibitor that induces apoptosis haemoglobin, or the antioxidants N-acetylcysteine, glutathione (Searle et al., 1975), was used as a positive control. and ascorbic acid. Time-course experiments with nitric oxide donors A freshly prepared solution of GSNO in 10% CM (1 mM) was added to the cell monolayers for defined incubation times from 0 to 24 hours. MATERIALS AND METHODS For SNAP experiments, a freshly prepared solution (1 mM) in 10% CM was added to quiescent cell monolayers at 24, 48 and 72 hour Materials intervals. Unless stated, all chemicals were purchased from Sigma (Poole, Dorset, UK) and used without further purification. Nitric oxide and sodium nitroprusside trapping experiments Preparation of aqueous solutions of nitric oxide The NO scavenger oxyhaemoglobin and the antioxidants ascorbic NO was generated by reacting concentrated H2SO4 with a saturated acid or N-acetylcysteine were each diluted in 10% CM (to the con- solution of sodium nitrite and purified by passing the resulting gas centrations specified in the figure legends) and preincubated with through 10 M NaOH and distilled H2O. Saturated solutions of NO either GSNO for 24 hours, or SNP for 2 hours. In studies using sodium were prepared by bubbling purified NO through deoxygenated nitroprusside (SNP), the antioxidant glutathione was also tested. Dulbecco’s modified Eagle’s medium (DMEM, ICN Biomedicals, These solutions were added to quiescent 3T3 cultures and incubated Thame, Oxon, UK) for 30 minutes at room temperature (20°C). The (37°C) for 2 or 24 hours. The 2 hour cultures were washed with pH of the medium was maintained between pH 7.2 and 7.4 by the serum-free DMEM and incubated for a further 22 hours with 10% CM addition of hydrochloric acid. The concentration of the saturated NO (0.5 ml/well). In a separate series of experiments, defined antioxidants solution at 20°C is known to be 1.7 mM and NO solutions were were added after the NO species. For NO, cultures were exposed to diluted in conditioned medium (CM, spent medium removed from GSNO for 8 hours and then washed and the medium replaced with quiescent monolayers) and DMEM to give a final solution of NO in 10% CM alone or containing 0.3 mM ascorbic acid. For NO+, ascorbic 10% CM at the concentrations specified in the figure legends. acid (0.3 mM) was added to Swiss 3T3 cells at defined time points before or after SNP addition (1 mM); exposure to SNP was for a total Synthesis of S-nitrosoglutathione and S-nitroso-N- of 2 hours. For each NO species, apoptosis was measured 24 hours acetylpenicillamine after the start of the experiment as described in the text. GSNO was prepared by the reaction of acidified sodium nitrite solution with glutathione. The resulting solution was treated with Fluorescence microscopy acetone to precipitate the GSNO, which was washed then dried in At the end of each experiment, cell cultures were fixed by the addition vacuo (Hart, 1985). SNAP was synthesised by the reaction of acidified of one volume of methanol:glacial acetic acid (3:1 v/v) for 3 minutes sodium nitrite solution with N-acetylpenicillamine. The resulting at room temperature. This was aspirated and replaced with the same green solid was washed and then dried in vacuo (Moynihan and fixative (0.5 ml/well) for a further 3 minutes. Cultures were then Roberts, 1994). washed twice with phosphate-buffered saline (PBS) and incubated with propidium iodide (PI, 60 µg/ml) in the presence of DNAse-free Preparation of oxyhaemoglobin RNAse (300 µg/ml) for 30 minutes at 37°C. PI-labelled nuclei were Oxyhaemoglobin was prepared according to the method of Ignarro et visualised with the aid of a Fluovert FU fluorescence microscope al. (1987). A solution of bovine crystalline haemoglobin in Krebs buffer (Leitz, Wetzlar, Germany) using a ×25 objective. Apoptosis was was reduced with sodium dithionite, with excess sodium dithionite scored by counting the proportion of small, pyknotic, highly fluor- being removed from the reaction by passage through a Sephadex G25 escent nuclei to normal, diffusely stained ones. A minimum of 200 column. The resulting solution was then exposed to air for 30 minutes. nuclei per field were counted, with at least six fields for each experi- The concentration of oxyhaemoglobin was determined by spectrome- mental point. try as described previously (Feelisch and Noack, 1987) and diluted in 10% CM to the concentrations specified in the figure legends. Electron microscopy Following treatment with NO or NO+ donors for 2 (NO+) or 16 (NO) Cell culture hours Swiss 3T3 cells (grown on coverslips on 24-well plates) were Swiss 3T3 fibroblasts were seeded at a density of 5×104 cells/dish fixed in 2% glutaraldehyde for 2 hours at 4°C, washed, osmicated and onto 90 mm tissue culture dishes (Nunc, Roskilde, Denmark) in 10 dehydrated before embedding in Taab® resin.