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Originally published In Press as doi:10.1074/jbc.M111177200 on February 22, 2002

J. Biol. Chem., Vol. 277, Issue 18, 16067-16074, May 3, 2002
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Hypoxia Sensitizes Cells to Nitric Oxide-induced Apoptosis*

Vivian Y. Lee, David S. McClintock, Matthew T. Santore, G. R. Scott Budinger, and Navdeep S. ChandelDagger

From the Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611

Received for publication, November 21, 2001, and in revised form, February 11, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O2) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-XL protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax-/- bak-/- mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho °-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) is a diffusible messenger that has been implicated in numerous physiological and pathological conditions (1, 2). Classical studies revealed that biological activity of NO was to regulate blood flow by relaxing the vasculature. Nitric oxide-induced vasodilation occurs by activating a guanylate cyclase-initiated signaling cascade (3). Recent studies have described NO as effecting a variety of other cellular functions through direct posttranslational modifications of proteins directly (i.e. nitrosylation and nitration), reacting with reactive oxygen species, and interacting with proteins that contain a heme moiety (4). The mitochondrial enzyme cytochrome c oxidase is one particular heme protein with which NO interacts, resulting in the inhibition of the enzymatic activity of the oxidase (5, 6). Under normal physiologic conditions, cytochrome c oxidase is the final electron acceptor of the respiratory chain, resulting in the reduction of oxygen to water. NO acts as a competitive inhibitor with respect to oxygen, and NO inhibition of cytochrome c oxidase has been shown to regulate cellular respiration and apoptosis under normal oxygen conditions (7-11).

NO inhibition of cytochrome c oxidase induces apoptosis by causing a decrease in mitochondrial membrane potential and cytochrome c release from mitochondria (12, 13). Cytochrome c released into the cytoplasm can directly interact with apoptotic protease activating factor (Apaf-1),1 leading to the activation of caspase-9 (14, 15). Activated caspase-9 can activate caspases-3 and -7, resulting in the morphological features of apoptosis. Anti-apoptotic members such as Bcl-2 and Bcl-XL inhibit cell death by preventing the release of cytochrome c, whereas pro-apoptotic Bcl-2 family members such as Bax or Bak are sufficient to trigger the release of cytochrome c, resulting in apoptosis (for review see Ref. 16). Furthermore, Bax or Bak are required for mitochondrial-dependent apoptosis in response to a variety of death stimuli, including growth factor withdrawal (17, 18). Bcl-XL has been shown to prevent NO-induced apoptosis, whereas Bax has been hypothesized to mediate NO-induced apoptosis (1, 19, 20). The interaction of NO with superoxide resulting in the formation of peroxynitrite (ONOO-) is another proposed mechanism by which NO induces apoptosis (1, 21, 22). Peroxynitrite causes cell death by irreversibly inhibiting mitochondrial electron transport (23-26) and by opening the mitochondrial permeability transition pore (27-29). Most previous studies have examined the effects of NO on cell death during 21% O2. Because NO is a competitive inhibitor of cytochrome c oxidase with respect to oxygen, in the present study we examined the effect of NO on cell survival under normoxia (21% O2) and hypoxia (1.5% O2).

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Rat1a fibroblasts were cultured at 30-50% confluence in Dulbecco's modified essential medium supplemented with HEPES (10 mM), pyruvate (1 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% heat-inactivated fetal bovine serum (Invitrogen). Rat1a fibroblasts transfected with Bcl-XL and the control neomycin vector were gifts of Dr. Nissim Hay (30). Mouse embryonic fibroblasts generated from wild-type or bax-/-bak-/- embryos were a gift from Dr. Craig Thompson (18). Wild-type HT1080 fibrosarcoma cells were incubated in Dulbecco's modified essential medium containing ethidium bromide (100 ng/ml), sodium pyruvate (1 mM), and uridine (100 µg/ml) to generate Á°-HT1080 cells (31). The rho ° status of cells was confirmed by the absence of cytochrome oxidase subunit II by polymerase chain reaction and by the failure to grow in the absence of uridine in the medium. Hypoxic conditions (1.5% O2, 93.5% N2, and 5% CO2) were achieved in a humidified variable aerobic work station (INVIVO O2, Ruskinn Technologies) at 37 °C. Nitric oxide was added to cells using the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2 diolate (DETA-NO, Alexis Corp.).

Measurement of Cell Death-- Cell death was assayed by measuring lactate dehydrogenase activity (LDH) in culture supernatants using a cytotoxicity detection kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. Apoptosis was detected as a percentage of cells, which had condensed, or by fragmented nuclei with Hoechst 33258 staining (1 µg/ml, Sigma) as described previously (32).

Cytochrome c Immunostaining-- Rat1a fibroblasts were plated on 60-mm culture dishes at 20-30% confluence and exposed to experimental conditions. Both adherent and non-adherent cells were washed with phosphate-buffered saline and placed on a glass slide at 14,000 × g for 5 min (Cytospin®3 Cytocentrifuge, Thermo Shandon). The cells were fixed with 40% methanol (5 min, -20 °C), blocked in 1% bovine serum albumin (Sigma), and incubated for 2 h with 1 µg/ml of anti-cytochrome c monoclonal antibody (BD PharMingen) at 37 °C in a humidified environment. The cells were then washed in phosphate-buffered saline containing 0.1% bovine serum albumin, air-dried, and incubated for 1 h with 1 µg of rhodamine-conjugated secondary antibody (Chemicon International). Subsequently, the cells were washed as before, air-dried, and stained with 4',6-diamidino-2-phenylindole (DAPI)/1,4-diazabicyclo-[2.2.2]octane (DABCO).

Measurement of Caspase Activity-- Caspase-9 enzymatic activity was measured with fluorometric assay kits specific to each caspase (R&D Systems). Cell were plated onto 100-mm culture dishes at 40-60% confluence, and caspase activity was measured according to the manufacturer's protocol using a fluorescent microplate reader. Data were normalized using total protein concentration as determined by the Bio-Rad protein assay (Bio-Rad).

Measurement of Mitochondrial Membrane Potential (psi )-- To assess the Delta psi , cells were plated onto 60 mm culture dishes at 40-60% confluence and incubated 1 h prior to the time point in the presence of two fluorescence probes, tetramethylrhodamine ethyl ester (TMRE, excitation, 550 nm; emission, 580 nm; at 200 nM) and Mitotracker Green (MITO; excitation, 490 nm; emission, 515 nm; at 1 µM) (Molecular Probes). Cells were lysed with 1% (v/v) Triton X-100, and fluorescence was measured on a SpectraMax Gemini microplate reader (Molecular Devices). TMRE localizes within the mitochondria, and its fluorescence increases in proportion to the mitochondrial membrane potential. Mitotracker Green fluorescence localizes to the mitochondria independently of Delta psi and reflects the number of mitochondria within a given cell. The ratio between TMRE fluorescence and MITO fluorescence reflects mitochondrial membrane potential normalized to the number of mitochondria. As a control for each condition, cells were incubated with both TMRE and MITO in the presence of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 20 µM) (Sigma), which dissipates the psi . For each condition, the ratio of TMRE and MITO was subtracted from the TMRE/MITO ratio in the presence of FCCP [(TMRE/MITO) - (TMRE/MITO)FCCP].

Statistical Analysis-- The data presented are the mean ± S.E. of four independent experiments. Data were analyzed using one-way analysis of variance. Statistical significance was determined at the 0.05 level.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia Sensitizes Cells to Nitric Oxide-induced Cell Death-- Rat1a fibroblasts were treated with 0 and 100 µM DETA-NO for 24 and 48 h under normoxia (21% O2) or hypoxia (1.5% O2), and cell death was determined by LDH release. At 24 h, Rat1a fibroblasts exposed to 100 µM DETA-NO under 1.5% O2 displayed similar cell death as cells under 21% O2 (Fig. 1A). In contrast, at 48 h, Rat1a fibroblasts exposed to 100 µM NO under 1.5% O2 displayed a significantly increased cell death compared with cells treated with 100 µM NO under 21% O2 (Fig. 1A). To determine whether DETA-NO-mediated effects on cell death were through the generation of nitric oxide, Rat1a fibroblasts were exposed to 0 or 100 µM DETA-NO under 1.5% O2 in the presence of hemoglobin, a nitric oxide scavenger. Fig. 1B illustrates that hemoglobin prevents DETA-NO-induced cell death during hypoxia. To determine whether the cell death during hypoxia in the presence of DETA-NO was apoptotic, condensed and fragmented nuclei were examined with Hoechst staining in Rat1a fibroblasts exposed to 0 or 100 µM DETA-NO under 1.5% and 21% O2 for 48 h. Rat1a fibroblasts displayed a significant increase in apoptotic nuclei in the presence of 100 µM DETA-NO under 1.5% O2 compared with the cells exposed to 100 µM DETA NO under 21% O2 (Fig. 1, C and D). Taken together, these data demonstrate that prolonged hypoxia sensitizes cells to nitric oxide-induced apoptosis.


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  		Fig. 1.   A, Rat1a fibroblasts were treated with 0 and 100 µM DETA-NO under 21% and 1.5% O2 for 24 or 48 h, and cell death was assessed by LDH release. B, Rat1a fibroblasts were treated with 0 and 100 µM DETA-NO under 1.5% O2 for 48 h in the presence of hemoglobin, and cell death was assessed by LDH release. C, the percentage of apoptotic cells scored by Hoechst staining of Rat1a fibroblasts exposed to 0 or 100 µM DETA-NO under 21% and 1.5% O2 for 48 h is shown. D, apoptotic cells scored by Hoechst staining of Rat1a fibroblasts exposed to 0 µM or 100 µM DETA-NO under 21% and 1.5% O2 for 48 h are shown. *, p < 0.05 compared with cells exposed to 0 µM DETA-NO under 21% O2.

Bcl-2 Family Members Regulate NO-induced Apoptosis during Hypoxia-- Rat1a fibroblasts stably transfected with the control vector (neomycin) or Bcl-XL were treated with 0 or 100 µM DETA-NO under 21 and 1.5% O2 for 48 h, and cell death was determined by LDH release. DETA-NO-treated cells transfected with Bcl-XL did not display increased cell death under 1.5% O2 compared with 21% O2 (Fig. 2A). In contrast, DETA-NO-treated neomycin control cells displayed an increase in cell death during 1.5% O2 compared with 21% O2 (Fig. 2A). To determine the role of Bax or Bak in nitric oxide-induced cell death during hypoxia, murine embryonic fibroblasts from wild-type or bax-/- bak-/- were exposed to 0 µM or 100 µM DETA-NO under 21 and 1.5% O2 for 24 h and assessed for cell death by LDH release. Wild-type murine embryonic fibroblasts were sensitive to NO-induced death during hypoxia (Fig. 2B). In contrast, bax-/- bak-/- murine embryonic fibroblasts were resistant to NO-induced cell death during hypoxia. These results indicate that nitric oxide-induced cell death during hypoxia requires Bax or Bak and that cell death is prevented by Bcl-XL.


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  		Fig. 2.   A, Rat1a fibroblasts were stably transfected with the control vector neomycin (Neo) or Bcl-XL treated with 0 or 100 µM DETA-NO under 21% and 1.5% O2 for 48 h and assessed for cell death by LDH release. B, wild-type and bax-/- bak-/- murine embryonic fibroblasts were exposed to 0 or 100 µM DETA-NO under 21 and 1.5% O2 for 48 h and assessed for cell death by LDH release. *, p < 0.05 compared with neomycin control or wild-type cells exposed to 0 µM DETA-NO under 21% O2.

NO-induced Cell Death Is Accompanied by Cytochrome c Release-- To determine how long cells could survive nitric oxide exposure under 1.5% O2 before committing to apoptosis, neomycin control cells were exposed to 100 µM DETA-NO under 1.5% O2 for various time intervals. Subsequently, the DETA-NO was removed, and the cells were allowed to recover under 1.5% O2 for a total of 48 h. Neomycin control cells exposed to 100 µM DETA-NO for 9 h (followed by a 39-h incubation with 0 µM DETA-NO) did not display a significant cell death compared with cells incubated with 0 µM DETA-NO for the 48 h duration. However, cells exposed to 100 µM DETA-NO for 18 h followed by 30 h incubation with 0 µM DETA-NO did display a significant cell death compared with cells incubated with 0 µM DETA-NO for the 48 h duration. These results indicate that the commitment to NO-induced cell death under 1.5% O2 occurs between 9 and 18 h of exposure to NO (Fig. 3A).


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  		Fig. 3.   A, Neomycin (Neo) control cells were exposed to either 0 µM DETA-NO (1) or 100 µM DETA-NO (4) for 48 h. In a separate series of experiments, cells were exposed to 100 µM DETA-NO under 1.5% O2 for either 9 (2) or 18 h (3), after which the DETA-NO was removed with cell death measured at 48 h by LDH release. B, neomycin control and Bcl-XL cells were treated with 100 µM DETA-NO under 1.5% O2, and cytochrome c release was assessed by immunostaining. *, p < 0.05 compared with neomycin control exposed to 0 µM DETA-NO under 21% O2.

To examine whether the commitment to die coincided with the release of cytochrome c, neomycin control and Bcl-XL cells were exposed to 0 and 100 µM DETA-NO under 21 and 1.5% O2. Mitochondrial cytochrome c release was examined at 9 and 18 h using a fluorescence-tagged anti-cytochrome c antibody. Visualizing the cells under fluorescent microscopy, cytochrome c localized to the mitochondria displays a punctate pattern, whereas cytochrome c localized to the cytoplasm displays a diffuse pattern. Neither neomycin nor Bcl-XL cells released cytochrome c after 9 h with 100 µM DETA-NO under 1.5% O2 compared with cells exposed to 0 µM DETA-NO (data not shown). After 18 h of exposure to 100 µM DETA-NO under 1.5% O2, neomycin control cells had released cytochrome c into the cytoplasm, whereas Bcl-XL cells maintained cytochrome c within the mitochondria (Fig. 3B). Cytochrome c, following release from the mitochondria, has been shown to bind to Apaf-1, which then undergoes a conformational change that allows the cleavage and activation of caspase-9 (14, 15). Neomycin control cells displayed a significantly greater -fold induction of caspase-9 compared with Bcl-XL cells after being exposed to 18 h of 100 µM DETA-NO at 1.5% O2 (Fig. 4A). Previous studies suggest that the release of cytochrome c is accompanied by changes in the mitochondrial membrane potential (33). Neomycin control and Bcl-XL cells were exposed to 100 µM DETA-NO under 1.5% O2 for 18 h, and inner mitochondrial membrane potential psi  was measured using the ratio between TMRE and MITO. In response to 100 µM DETA-NO and 1.5% O2, neomycin control cells at 18 h exhibited a significantly greater psi  depolarization compared with Bcl-XL cells (Fig. 4B). These data indicate that the commitment to death in response to NO during hypoxia is at the point of cytochrome c release and caspase-9 activation.


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  		Fig. 4.   A, neomycin (Neo) control- and Bcl-XL-transfected cells were exposed to 0 and 100 µM DETA-NO under 1.5% O2, and caspase-9 enzymatic activity was measured. Data are normalized to the neomycin control cells at 0 µM DETA-NO and 1.5% O2. B, neomycin control- and Bcl-XL-transfected cells were exposed to 100 µM DETA-NO under 1.5% O2, and mitochondrial membrane potential was measured at 18 h. Data are normalized to the neomycin control cells at 0 µM DETA-NO. *, p < 0.05 compared with neomycin control cells exposed to 0 µM DETA-NO under 21% O2.

Bcl-XL Requires Both Glycolysis and F1F0-ATP Synthase to Prevent Nitric Oxide-induced Cell Death under Hypoxia-- Prior studies speculate that the anti-apoptotic properties of Bcl-XL are attributed to the ability of this protein to maintain mitochondrial membrane homeostasis (33, 34). Furthermore, studies have suggested that cells undergoing electron transport inhibition by nitric oxide can continue to maintain a mitochondrial membrane potential through the utilization of glycolytic ATP by the reversal of the F1F0 ATP synthase (11). To determine whether functional glycolysis and a functional F1F0 ATP synthase are required for Bcl-XL to prevent nitric oxide-induced cell death under hypoxia, neomycin control and Bcl-XL cells were incubated for 24 h with 100 µM DETA-NO under 1.5% O2 in the presence and absence of the glycolytic inhibitor 2-deoxyglucose (2-DOG, 12 mM) and the F1F0 ATP synthase inhibitor, oligomycin (5 µM). Oligomycin and 2-DOG both independently caused cell death in neomycin control cells but not in Bcl-XL cells under normal oxygen conditions in the absence of DETA-NO (data not shown). Neomycin and Bcl-XL cells treated with nitric oxide during hypoxia underwent a non-apoptotic form of cell death in the presence of 2-DOG (Fig. 5). By contrast, Bcl-XL cells treated with nitric oxide during hypoxia underwent an apoptotic form of cell death in the presence of oligomycin (Fig. 6). These data suggest that glycolysis and a functional F1F0 ATP synthase are required for Bcl-XL to prevent nitric oxide-induced cell death under hypoxia.


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  		Fig. 5.   A, neomycin (Neo) control and Bcl-XL cells were incubated for 24 h with 100 µM DETA-NO under 1.5% O2 in the presence and absence of the glycolytic inhibitor 2-deoxyglucose (2-DOG, 10 mM), and cell death was assessed by LDH release. B, percentage of apoptotic cells scored by Hoechst staining of neomycin control and Bcl-XL cells exposed to 100 µM DETA-NO under 1.5% O2 in the presence of 2-DOG for 24 h is shown.


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  		Fig. 6.   A, Bcl-XL cells were treated for 24 h with 100 µM DETA-NO under 1.5% O2 in the presence or absence of the F1F0 ATP synthase inhibitor oligomycin (Oligo, 5 µg/ml). B, percentage of apoptotic cells scored by Hoechst staining of Bcl-XL cells exposed to 100 µM DETA-NO under 1.5% O2 in the presence of oligomycin (5 µg/ml) for 24 h is shown.

NO-induced Cell Death during Hypoxia Is Independent of cGMP or Peroxynitrite-- Previous studies have demonstrated that nitric oxide can combine with superoxide to generate the toxic free radical peroxynitrite (ONOO-) or activate guanylate cyclase to generate cGMP (3, 35). Both peroxynitrite and cGMP have been implicated in NO-induced apoptosis (21-26). To determine whether the NO-induced death under 1.5% O2 was due to cGMP or peroxynitrite, Rat1a fibroblasts were exposed to 0 and 100 µM DETA-NO under 1.5% O2 for 48 h with the cGMP inhibitor H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) at 10 µM, the peroxynitrite scavenger ebselen at 10 µM, or the superoxide scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin (TBAP) at 150 µM. Although ODQ, ebselen, and TBAP had no significant cytotoxic effects, these compounds also did not have any protective effects against nitric oxide-induced cell death during hypoxia (Fig. 7). Ebselen and TBAP did prevent SIN-1-induced cell death. SIN-1 releases both superoxide and nitric oxide, thus generating peroxynitrite (Fig. 7). This suggests that the increase in nitric oxide-induced cell death under hypoxia is independent of cGMP activity and peroxynitrite.


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  		Fig. 7.   Left panel, Rat1a fibroblasts were treated for 48 h with 0 and 100 µM DETA-NO under 1.5% O2 in the presence and absence of the peroxynitrite scavenger ebselen (EBS, 10 µM), the cGMP inhibitor H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), and the superoxide scavenger TBAP (150 µM). Right panel, Rat1a fibroblasts were treated for 24 h with 75 µM SIN-1 under 21% O2 in the presence of ebselen (10 µM) or TBAP (150 µM). *, p < 0.05 compared with cells exposed to 0 µM DETA-NO or SIN-1 under 21% O2.

Nitric Oxide-induced Cell Death during Hypoxia Requires a Functional Electron Transport Chain-- A possible mechanism for the increase in nitric oxide-induced cell death under hypoxia is the direct inhibition of mitochondrial electron transport by nitric oxide. ATP levels were measured to examine whether electron transport was inhibited during hypoxia in the presence of nitric oxide. ATP levels decreased in both Neomycin and Bcl-XL cells in the presence of 100 µM DETA-NO during hypoxia (Fig. 8). Oligomycin did not alter the ATP levels in neomycin or Bcl-XL cells in the presence of 100 µM DETA-NO during hypoxia, indicating that mitochondrial ATP generation was maximally inhibited by NO during hypoxia (Fig. 8). In contrast, 2-DOG completely abolished ATP levels in both neomycin and Bcl-XL cells in the presence of 100 µM DETA-NO during hypoxia (Fig. 8). Thus, NO inhibits the generation of mitochondrial ATP during hypoxia, and glycolysis is the main source of ATP in these cells.


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  		Fig. 8.   Neomycin (NEO) control cells (A) and Bcl-XL cells (B) were exposed to 0 µM or 100 µM DETA-NO under 1.5% O2 for 8 h in the presence of oligomycin (OLIGO, 5 µg/ml) or 2-deoxyglucose (2-DOG, 10 mM), and the levels of ATP were measured. Data are normalized to the 0 µM DETA-NO under 1.5% O2.

To further elucidate the role of functional mitochondrial electron transport in nitric oxide-induced cell death during hypoxia, HT1080 human fibrosarcoma cells containing cytochrome c but lacking a functional mitochondrial electron transport chain (rho °-cells) were exposed to 0 or 100 µM DETA-NO under 21 and 1.5% O2 for 48 h and assessed for cell death by LDH release. Wild-type HT1080 cells displayed increased cell death in response to nitric oxide under hypoxia compared with normoxia, whereas rho °-cells did not show an increase in nitric oxide-induced cell death under hypoxia (Fig. 9). These results suggest that NO-induced cell death under 1.5% O2 requires a functional mitochondrial electron transport chain.


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  		Fig. 9.   Wild-type HT1080 human fibrosarcoma cells and HT1080 cells lacking a functional mitochondrial electron transport chain (rho °-cells) were exposed to 0 µM or 100 µM DETA-NO under 21 and 1.5% O2 for 48 h and assessed for cell death by LDH release. *, p < 0.05 compared with wild-type cells exposed to 0 µM DETA-NO under 21% O2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide has been implicated in cellular injury and death in a variety of cells. Previous studies have examined NO-mediated cell death under normal oxygen conditions (1, 3). In the present study, we examined whether hypoxia (1.5% O2) sensitized Rat1a fibroblasts to NO-induced cell death. DETA-NO administered at 100 µM was non-toxic under 21% O2. However, at this concentration cells underwent death under hypoxic conditions. NO-induced cell death under hypoxia is apoptotic and is preceded by the release of cytochrome c from the mitochondria and the activation of caspase-9. In cells exposed to hypoxia in the presence of nitric oxide, the commitment to death occurred at the point when cells released cytochrome c and activated caspase-9. The loss of cytochrome c was accompanied by the dissipation of the mitochondrial membrane potential. Bcl-XL, an anti-apoptotic protein that localizes to the outer mitochondrial membrane, was able to prevent the dissipation of mitochondrial membrane potential, the release of cytochrome c, caspase-9 activation, and cell death during hypoxia in the presence of NO. The ability of Bcl-XL to prevent apoptosis is attributed to its ability to negate the effects of pro-apoptotic Bcl-2 family members (17, 18). Thus, either the overexpression Bcl-XL or the loss of Bax and Bak prevents apoptosis in response to a variety of death stimuli by inhibiting the release of cytochrome c. Indeed, we found that fibroblasts lacking Bax and Bak were resistant to nitric oxide-induced death during hypoxia. Taken together, our current observations suggest that Bcl-2 family members also regulate nitric oxide-induced apoptosis during hypoxia.

Nitric oxide can combine with superoxide to form peroxynitrite, which can induce apoptosis by irreversibly inhibiting mitochondrial complex I, II, or V (21). Previous studies have indicated that hypoxia can increase the generation of superoxide from mitochondrial complex III within minutes (36). One mechanism by which nitric oxide initiates cell death under hypoxia could be an irreversible inhibition of electron transport chain by peroxynitrite. However, our current data indicate that neither ebselen (peroxynitrite scavenger) nor TBAP (superoxide scavenger) prevented nitric oxide-induced cell death under hypoxia. Ebselen and TBAP did prevent SIN-1 (peroxynitrite generator)-induced cell death. Both ebselen and TBAP independently have been shown to inhibit peroxynitrite-mediated cell death in a variety of cells (37-39). Thus, peroxynitrite generation is not likely to be involved in initiating nitric oxide-induced cell death under hypoxia.

It is well established that nitric oxide is a reversible competitive inhibitor of cytochrome c oxidase with respect to oxygen (5, 6). The affinity of cytochrome c oxidase for nitric oxide increases as oxygen tensions are decreased, resulting in the inhibition of cellular respiration, which reverses upon nitric oxide removal. Our current data demonstrate that cells exposed to 9 h of hypoxia in the presence of NO followed by 39 h of hypoxia alone did not undergo cytochrome c release or subsequent cell death. By contrast, cells exposed to NO for 18 h under hypoxic conditions had released cytochrome c and thus were committed to apoptosis. These data suggest that the effects of NO during hypoxia prior to cytochrome c release are reversible. Furthermore, ATP levels decreased during hypoxia in the presence of NO. Oligomycin, an inhibitor of the F1F0-ATP synthase that prevents the formation of mitochondrial generated ATP, did not further decrease ATP levels during hypoxia in the presence of NO. Therefore, NO inhibits the generation of mitochondrial ATP during hypoxia. The remaining ATP in cells exposed to NO during hypoxia was abolished by 2-DOG, indicating that glycolysis is the main source of ATP. The role of electron transport inhibition as the primary mechanism was further supported by the observation that rho °-cells, cell that lack the catalytic subunits of cytochrome c oxidase but still contain a functional cytochrome c, failed to undergo cell death during hypoxia in the presence of NO. Previous studies have shown that rho °-cells are susceptible to cell death in response to traditional apoptotic stimuli such as growth factor withdrawal and staurosporine, yet are resistant to cell death induced by electron transport inhibitors (40). Collectively, our findings suggest that NO induces cell death during hypoxia by inhibiting the electron transport chain at the level of cytochrome c oxidase.

If the mechanism of nitric oxide-induced cell death under hypoxia was due to a reversible inhibition of mitochondrial electron transport, then how does an outer mitochondrial membrane protein like Bcl-XL act to protect cells against cell death? A simple explanation is that Bcl-XL suppresses the apoptotic actions of Bax or Bak. However, cells would still have to maintain their mitochondrial membrane potential in the absence of a functioning electron transport chain during hypoxia in the presence of nitric oxide. Under normal physiological conditions, electron transfer through the respiratory chain is coupled to the directional movement of protons across the inner mitochondrial membrane. This movement across the membrane establishes a pH gradient and a membrane potential that provides the thermodynamic driving force for F1F0-ATP synthase to generate ATP in the matrix. Recently, it was demonstrated that nitric oxide-induced inhibition of electron transport under normal oxygen tensions resulted in the hydrolysis of glycolytic ATP by F1F0-ATP synthase, which extrudes protons from the matrix in an attempt to maintain mitochondrial membrane potential (11). Bcl-XL was not able to prevent cell death in the presence of either a glycolytic inhibitor or a F1F0 ATP synthase inhibitor. We speculate that Bcl-XL prevents Bax or Bak from initiating cytochrome c release, thus allowing glycolysis and F1F0 ATP synthase to maintain mitochondrial homeostasis.

In summary, our present data demonstrate that low oxygen tensions sensitize cells to nitric oxide-induced cell death through a mechanism dependent on pro-apoptotic Bcl-2 family members and the inhibition of functional electron transport. Limited exposure of cells to nitric oxide under hypoxia may allow for full cell recovery and viability, with mitochondrial membrane homeostasis being maintained through utilization of glycolytic ATP. By contrast, extended continuous exposure to nitric oxide activates mitochondrial-dependent apoptosis. Further studies examining the mechanisms underlying increased nitric oxide-induced cell death under hypoxia may give us insight into the pathogenesis of cell death in the setting of excess nitric oxide production and hypoxia as exists in sepsis-related organ damage and dysfunction.

    FOOTNOTES

* This work was supported by the Crane Asthma Center, National Institutes of Health Grant GM60472-02 (to N. S. C.), and National Institutes of Health National Research Service Award HL68456-01 (to V. Y. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Division of Pulmonary and Critical Care, Tarry Building 14-707, 303 East Chicago Ave., Chicago, IL 60611-3010. Tel.: 312-503-2549; Fax: 312-908-4650; E-mail: nav@northwestern.edu.

Published, JBC Papers in Press, February 22, 2002, DOI 10.1074/jbc.M111177200

    ABBREVIATIONS

The abbreviations used are: Apaf-1, apoptotic protease activating factor; LDH, lactate dehydrogenase; TMRE, tetramethylrhodamine ethyl ester; MITO, Mitotracker Green; DETA-NO, (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2 diolate; 2-DOG, 2- deoxyglucose; TBAP, Mn(III)tetrakis(4-benzoic acid)porphyrin; SIN-1, 3-(4-morpholinyl)-sydnoneimine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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