From the Department of Medicine, University of
Massachusetts Medical School, Worcester, Massachusetts 01605 and
§ Department of Pediatrics, University of Virginia Medical
School, Charlottesville, Virginia 22908
Received for publication, December 6, 2002, and in revised form, March 14, 2003
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ABSTRACT |
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Cytochrome c released from
mitochondria into the cytoplasm plays a critical role in many forms of
apoptosis by stimulating apoptosome formation and subsequent caspase
activation. However, the mechanisms regulating cytochrome c
apoptotic activity are not understood. Here we demonstrate that
cytochrome c is nitrosylated on its heme iron during
apoptosis. Nitrosylated cytochrome c is found
predominantly in the cytoplasm in control cells. In contrast, when
cytochrome c release from mitochondria is inhibited by
overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia
(Bcl)-2 or Bcl-XL, nitrosylated cytochrome c is
found in the mitochondria. These data suggest that during apoptosis,
cytochrome c is nitrosylated in mitochondria and then
rapidly released into the cytoplasm in the absence of Bcl-2 or
Bcl-XL overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell
lysates. Moreover, the inhibition of intracellular cytochrome
c nitrosylation is associated with a decrease in apoptosis,
suggesting that cytochrome c nitrosylation is a
proapoptotic modification. We conclude that nitrosylation of the heme
iron of cytochrome c may be a novel mechanism of apoptosis regulation.
Apoptosis is a cell death pathway that removes excess, damaged,
autoreactive, or infected cells from organisms. Apoptosis may be
triggered by extrinsic stimuli or by cell surface receptors such as
Fas. Fas is a member of the tumor necrosis receptor superfamily that
mediates apoptosis when cross-linked by Fas ligand or by Fas agonist
antibody (1-3). Fas cross-linking results in the formation of the
death-inducing signaling complex composed of Fas, the adapter
protein FADD/MORT1, and caspase-8 (4-8). Caspase-8, a member of the
caspase family of cysteine proteases, is activated upon association
with the death-inducing signaling complex (9) and subsequently
activates downstream caspase members including caspases-3, -6, and -7 (10-13). These "effector caspases" cleave cellular proteins such
as poly(ADP) ribose polymerase, leading to apoptotic cell death. In
addition to directly activating downstream caspases, activated
caspase-8 cleaves Bid, a member of the B cell lymphoma/leukemia
(Bcl)1-2 family of proteins
(14-16). Truncated Bid trans-locates to the mitochondria where it
induces the release of proapoptotic molecules from the mitochondrial
intermembrane space including cytochrome c and a
subpopulation of caspase zymogens.
Once released into the cytoplasm, cytochrome c plays a
critical role in apoptotic pathways by binding to and inducing
oligomerization of the protein Apaf-1. The redox activity of cytochrome
c is not required for this apoptotic activity, but certain
structural elements of the protein are necessary because apocytochrome
c, denatured cytochrome c, and enzyme-digested
cytochrome c have no proapoptotic activity (17, 18). After
cytochrome c induces oligomerization of Apaf-1, the oligomer
binds procaspase-9, leading to its autoactivation (19-22). The
cytochrome c/Apaf-1/caspase-9 complex is called the apoptosome. Activated caspase-9 in the apoptosome activates downstream caspases such as caspase-3, leading to further propagation of the
apoptotic cascade.
The Bcl-2 family of proteins prevent aberrant apoptosis by regulating
the release of cytochrome c and other proapoptotic proteins from the mitochondrial intermembrane space (15, 18, 23-25). Anti-apoptotic family members such as Bcl-2 and Bcl-XL
inhibit, whereas proapoptotic members such as Bax and Bid stimulate the release of cytochrome c. The mechanisms by which Bcl-2
family members regulate the release of mitochondrial proteins are
controversial but may involve the formation of supramolecular openings
in the outer mitochondrial membrane (26).
Nitric oxide (NO) is a free radical gas that provides another level of
apoptosis regulation. The effects of NO on apoptosis are complex and
may be either stimulatory or inhibitory (27). One of the mechanisms by
which NO regulates biological processes including apoptosis is
nitrosylation of proteins (28). Nitrosylation is a posttranslational
modification involving the attachment of a NO group to a cysteine or
transition metal. Although the function of many proteins can be
modified by nitrosylation of critical cysteine residues and/or
transition metals in vitro, only a limited number of
proteins have been found to be endogenously nitrosylated in
vivo (28). We have previously shown that endogenous nitrosylation of the catalytic site cysteine of a subset of caspase members serves as
an on/off switch regulating caspase activity during apoptosis (29, 30).
The subset of caspases regulated by S-nitrosylation resides
predominantly in the mitochondria (30). This observation raised the
possibility that mitochondria are a key site of protein nitrosylation
in cells. To test this hypothesis, in the current studies we examined
whether cytochrome c, another mitochondrial protein that
plays a key role in apoptosis, is regulated by nitrosylation.
Cytochrome c has no free cysteines, and therefore, its heme
iron is the most probable nitrosylation target. Cytochrome c
can been heme nitrosylated in vitro (31-33) but has never
been shown to be nitrosylated intracellularly. Indeed, guanylate
cyclase and cytochrome oxidase are the only proteins whose function
clearly has been demonstrated to be regulated by endogenous heme
nitrosylation (34-38). The hemes of guanylate cyclase and cytochrome
oxidase are 5-coordinate and bind NO much more rapidly than the
6-coordinate heme of cytochrome c (39). Therefore, it is
unclear whether cytochrome c nitrosylation occurs
intracellularly. However, cytochrome c undergoes a
conformational change during apoptosis that may result in a
5-coordinate heme (40). This conformational alteration may increase the
reactivity of cytochrome c with NO. To further explore these
possibilities, in the current studies we determined whether 1)
cytochrome c is heme-nitrosylated intracellularly during apoptosis, and 2) if so, whether nitrosylation regulates cytochrome c function during apoptosis.
Cell Culture and Immunoprecipitation--
Cell lines were grown
at 37 °C, 5% CO2 in RPMI 1640 medium supplemented with
10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Logarithmically, growing cells were stimulated with Fas agonist antibody (clone CH-11,
60-240 ng/ml, Upstate Biotechnology). In some experiments, cells were
pretreated with
NG-monomethyl-L-arginine
(L-NMA) (5 mM versus 1 mM L-arginine in the medium) prior to
Fas stimulation. At various time points before and after Fas
stimulation, mitochondrial and cytoplasmic fractions were prepared as
described previously (30), except in some experiments cells were
homogenized in buffer containing 0.25 M sucrose, 5 mM Tris, pH 7.5, 50 mM KCl, 1 mM
EDTA, pH 8, 2 mM MgCl2, and protease inhibitors. Immunoprecipitation of mitochondrial and cytoplasmic fractions was performed as described previously (29, 30) using 10 µg
of an anti-cytochrome c monoclonal antibody (BD Transduction Laboratories) or equal concentrations of an isotype-matched IgG1 control antibody (Sigma). The immunoprecipitated proteins were separated on a 12% SDS-PAGE gel and visualized using a silver staining
kit as per the manufacturer's instructions (Sigma) or analyzed by
cytochrome c immunoblot using an anti-cytochrome
c monoclonal antibody (BD Transduction Laboratories), a
peroxidase-conjugated secondary antibody (Amersham Biosciences) and a
chemiluminescence detection system per the manufacturer's instructions
(Amersham Biosciences). Cytochrome c or bovine serum albumin
standards were used to quantitate the amount of cytochrome c
in each immunoprecipitate.
Nitrosylation Measurements--
Nitrosylation of proteins was
determined by chemical reduction/chemiluminescence as described
previously (41). Immunoprecipitated proteins were eluted from protein
G-Sepharose beads (Amersham Biosciences) in 200 µl of 100 mM glycine, pH 3. The eluted proteins (50 µl) were
injected into a 5-ml anaerobic solution containing 100 µM
CuCl, 100 µM cysteine, and 0.01% antifoam (pH 3, 50 °C) purged continuously with argon in a Sievers 280 nitric oxide
analyzer. In some experiments, 45 mM potassium
iodide and 10 mM I2 in glacial acetic
acid were used for chemical reduction (42). NO evolved was measured by
chemiluminescence. Data were interpreted as raw photoelectric output
(integrated using Sievers software) and as absolute NO evolved using NO
standards generated by S-nitrosoglutathione or in
vitro nitrosylated cytochrome c.
In Vitro Nitrosylation of Cytochrome c--
Ferricytochrome
c (horse heart, Sigma) (100-200 µM) was
in vitro nitrosylated with DETA-NONOate (10 mM) in 0.1 M sodium phosphate buffer, pH 5, for
1 h in the dark at room temperature. Excess DETA-NONOate was
removed by two sequential acetone precipitations.
UV-visible Spectroscopy--
Spectrophotometric measurements
were carried out using a Beckman Coulter DU 640B Spectrophotometer.
Caspase-3 Activation Assays--
To assess caspase-3 activation
by cytochrome c, cytosolic extracts prepared as described
previously (43) were incubated for the indicated times at 30 °C with
1 mM dATP (Amersham Biosciences) alone or in combination
with 10 µM control or in vitro nitrosylated cytochrome c. After the incubation, caspase-3 activation in
the extracts was determined by immunoblot analysis using a
caspase-3-specific mouse monoclonal antibody or a rabbit polyclonal
that detects caspase-3 active fragments (BD Transduction Laboratories)
as described previously (29).
Analysis of Apoptosis by Acridine Orange Staining--
Cells
were pelletted, and 10 µl of cell slurry was mixed with 10 µl of
acridine orange (50 µg/ml) diluted in phosphate-buffered saline. The
percentage of cells with apoptotic morphology (nuclear and cytoplasmic
condensation and nuclear fragmentation) was then analyzed on a wet
mount slide using a Nikon Eclipse TE200 fluorescent microscope. At
least 200 cells in three separate fields were counted for each measurement.
Cytochrome c nitrosylation was analyzed during
Fas-induced apoptosis of human mononuclear cell lines. Cytochrome
c was purified by immunoprecipitation from mitochondrial and
cytoplasmic fractions of the human monocytic cell line U-937 or the
human T cell line CEM at 0, 1, and 2 h after
Fas-stimulation with agonist antibody. Cytochrome c was
efficiently immunoprecipitated with its specific antibody but not with
equal concentrations of isotype-matched control antibody (Fig.
1a). A silver stain analysis
revealed that immunoprecipitated cytochrome c was
full-length and that associated proteins did not significantly
contaminate the immunoprecipitates (Fig. 1a).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cytochrome c is nitrosylated
during Fas-induced apoptosis. a, Silver
stain of cytochrome c and control immunoprecipitates. U-937
cells stimulated with Fas agonist antibody for 0, 1, or 2 h were
separated into cytoplasmic (Cyto) or mitochondrial
(Mito) fractions. Cytochrome c (Cyt c)
or isotype-matched IgG1 control (Ig) immunoprecipitates were
obtained from each fraction and analyzed on a silver-stained
polyacrylamide gel. Bovine serum albumin standards (24 and 6 ng) were
used to quantitate the amount of cytochrome c in each
immunoprecipitate. Immunoglobulin heavy chain (HC), light
chain (LC), and cytochrome c are indicated.
Molecular masses (MW) are indicated on the right.
The gel is representative of eight independent experiments.
b, cytoplasmic cytochrome c is nitrosylated
1 h after Fas stimulation. The NO:cytochrome c
stoichiometry of cytochrome c immunoprecipitated from the
cytoplasm and mitochondria at 0, 1, and 2 h after Fas stimulation
is shown. The cytochrome c-specific NO content was
determined by subtracting the NO-derived chemiluminescence signal in
control immunoprecipitates (background) from the signal in
cytochrome c immunoprecipitates. The NO content was
normalized for cytochrome c protein concentration as
determined by silver stain analysis using known concentrations of
cytochrome c or bovine serum albumin standards. The data
represent the mean ± S.E. of 6-10 separate experiments in two
different cell lines (U-937 and CEM). *, p = 0.005 versus mitochondria at 1 h, paired t test,
n = 8. c, cytochrome c
nitrosylation is reversed by UV light. Cytochrome c
immunoprecipitates obtained from the cytoplasm 1 h after Fas
cross-linking were divided into two samples, one of which was treated
with UV light for 20 min. The NO chemiluminescence signal from
untreated ( ) or UV-treated (+) cytochrome c
immunoprecipitates is shown. The NO chemiluminescence signal in
arbitrary units is plotted on the y axis, and the time over
which the signal is measured is plotted on the x axis. The
area under the curve is proportional to the amount of NO in the sample.
The data are representative of three independent experiments.
d, nitrosylated cytochrome c is found
predominantly in the mitochondria in cells overexpressing Bcl-2 or
Bcl-XL. The stoichiometry of NO:cytochrome c was
determined as described in b using cells overexpressing
Bcl-2 (n = 3) or Bcl-XL (n = 3). The data represent the mean ± S.E. of 4-6 separate
experiments. *, p < 0.007 versus cytoplasm
at 1 h, paired t test, n = 6.
The extent of nitrosylation of immunoprecipitated cytochrome c was determined by chemical reduction/chemiluminescence as described previously (30, 41). In this method, NO is displaced from S-NO and from a subpopulation of metal-NO bonds (including the Fe-NO bond of cytochrome c) in a saturated copper/cysteine solution and is detected by chemiluminescence. Measurements obtained using the copper/cysteine method were confirmed using 45 mM potassium iodide and 10 mM I2 in glacial acetic acid for chemical reduction (data not shown) (42). An analysis of cytochrome c immunoprecipitates indicated that little if any mitochondrial cytochrome c was nitrosylated either before or after Fas stimulation (Fig. 1b). In contrast, cytochrome c released into the cytoplasm was nitrosylated (Figs. 1b and 3c). The stoichiometry of NO to cytochrome c was 0.6 in immunoprecipitates obtained from the cytoplasm 1 h after Fas stimulation, indicating that a significant portion of cytoplasmic cytochrome c is nitrosylated at this time point. Cytoplasmic cytochrome c nitrosylation decreased 2 h after Fas stimulation, suggesting that the protein may be initially nitrosylated and then denitrosylated (Fig. 1b). Similar results were obtained using staurosporine as an apoptotic stimulus (data not shown). Cytochrome c nitrosylation was eliminated by pretreating immunoprecipitates with UV light, which cleaves NO from metal-NO and S-NO bonds, for 10-20 min, indicating that the NO signal was derived from protein nitrosylation rather than from contaminating nitrite (Fig 1c). In addition, pretreatment of the immunoprecipitates with K3Fe(CN)6 (0.2 M for 30 min) (Fig 3c) but not with HgCl2, which selectively displaces NO from S-NO bonds (data not shown), decreased the NO signal, suggesting that cytochrome c is heme-nitrosylated. Finally, pretreatment of cells with the nitric-oxide synthase (NOS) inhibitor L-NMA decreased cytochrome c nitrosylation, indicating that cytochrome c is endogenously nitrosylated (Fig 3c).
To confirm that cytochrome c is nitrosylated on its heme
iron during apoptosis, we analyzed the UV-visible absorption spectra of
nitrosylated cytochrome c immunoprecipitates. In
vitro nitrosylation of ferricytochrome c results in an
iron-nitrosyl peak at 562 nm and a Soret shift from 405 to 411 (Fig.
2a) (31-33). Nitrosylated but
not non-nitrosylated immunoprecipitated cytochrome c had a red-shifted Soret peak at 411 nm, consistent with heme nitrosylation (Fig. 2b). The high concentrations of protein (~10
µM) required to visualize the 562 nM peak
precluded the analysis of this peak in our immunoprecipitated samples.
The red-shifted Soret peak of immunoprecipitated cytoplasmic cytochrome
c was not the result of cytochrome c reduction
(Fig. 2c), because the shift was stable at pH 3, whereas
reduced cytochrome c is oxidized at this pH, resulting in a
Soret peak shift back to 405 nm (Fig. 2d).
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It is possible that cytochrome c is directly nitrosylated in the cytoplasm or is nitrosylated in the mitochondria and then rapidly released into the cytoplasm. To distinguish between these possibilities, we determined whether nitrosylated cytochrome c is found in mitochondria when its release into the cytoplasm is inhibited by overexpression of the anti-apoptotic proteins, Bcl-2 or Bcl-XL (23). Nitrosylation of immunoprecipitated cytochrome c was analyzed in Fas-stimulated U-937 cells stably transfected with Bcl-2, Bcl-XL, or control expression vectors (44). In contrast to control cells, nitrosylated cytochrome c was found predominantly in the mitochondria 1 h after Fas stimulation in cells overexpressing Bcl-2 or Bcl-XL (Fig. 1d). These data suggest that cytochrome c is nitrosylated within mitochondria and then, in the absence of Bcl-2 and Bcl-XL overexpression, released into the cytoplasm.
Once released into the cytoplasm, cytochrome c forms a
complex with Apaf-1 and caspase-9 called the apoptosome that cleaves and activates downstream caspases such as caspase-3. To determine whether nitrosylation of cytochrome c alters its ability to
stimulate apoptosome formation and subsequent caspase-3 activation, we
analyzed caspase-3 activation in cytosolic extracts stimulated with
nitrosylated or control cytochrome c. The addition of
in vitro nitrosylated cytochrome c as compared
with equal concentrations of control cytochrome c to
cytoplasmic extracts increased caspase-3 activation (Fig.
3a). The levels of the p20
active fragment were increased at 60 min, and the levels of both the
p20 and p17 active fragments were increased after 120 min in lysates
stimulated with nitrosylated cytochrome c as compared
with control cytochrome c (Fig. 3a). These data
suggest that nitrosylation is a posttranslational modification that
enhances the proapoptotic function of cytochrome c, leading to increased caspase-3 activation.
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To further establish a causal relationship between cytochrome
c nitrosylation and apoptosis, we determined whether
inhibition of intracellular cytochrome c nitrosylation
decreased Fas-induced apoptosis. Pretreatment of CEM cells for 1 h
with the NOS inhibitor L-NMA abrogated Fas-induced
nitrosylation of cytoplasmic cytochrome c (Fig. 3,
b and c) and was associated with a decrease
in Fas-induced apoptosis (Fig. 3d). Thus, heme nitrosylation
of cytochrome c may stimulate Fas-induced apoptosis.
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DISCUSSION |
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In summary, our results suggest that nitrosylation of cytochrome c is a novel mechanism of apoptosis regulation in cells and a very early event in apoptotic signaling. The data indicate that apoptosis may be regulated in mammalian cells not only at the level of cytochrome c release from mitochondria but also by direct modification of cytochrome c. Because the heme edge of cytochrome c is involved in its association with Apaf-1 (45, 46), it is possible that heme nitrosylation of cytochrome c facilitates apoptosome formation and thereby stimulates caspase-3 activation. Ultimately, cytochrome c nitrosylation may provide a new therapeutic target for diseases associated with dysregulated apoptosis such as cancer, neurodegeneration, stroke, and autoimmunity.
The data provide one of the rare examples to date of endogenous heme nitrosylation regulating protein function. Guanylate cyclase and cytochrome oxidase are the other two proteins whose function has been clearly demonstrated to be regulated by endogenous heme nitrosylation (34-38). NO inhibits cytochrome oxidase activity by binding to the iron/copper binuclear center (36-38). NO activates guanylate cyclase by binding to the heme iron and inducing a conformational change in the enzyme (47). Nitrosylation may also be an allosteric regulator of cytochrome c function. However, our finding that cytochrome c is endogenously nitrosylated during apoptosis is unexpected because cytochrome c has a 6-coordinate heme that is significantly less reactive with NO than the 5-coordinate hemes of guanylate cyclase and cytochrome oxidase (39). One possible explanation for our results is that cytochrome c undergoes a subtle conformational change during apoptosis that increases the reactivity of the heme iron with NO. A number of investigators have demonstrated that cytochrome c undergoes a conformational change when bound to anionic phospholipid vesicles that model interactions of cytochrome c with the phospholipid-rich mitochondrial membranes (48-53). The conformational change involves an opening of the heme crevice, resulting in part from the loss of the iron-methionine 80 ligation. A similar conformational change in cytochrome c has been detected in cells early during apoptosis (40). The data raise the possibility that alterations in mitochondrial membrane lipids during apoptosis may induce a conformational change in cytochrome c, resulting in a 5-coordinate heme. Such a conformational alteration may facilitate stable heme nitrosylation of cytochrome c during apoptosis. Nitrosylation is unlikely to be the result of more drastic conformational changes such as enzymatic degradation or denaturation, because immunoprecipitated nitrosylated cytochrome c runs the same size as full-length cytochrome c on gels and the antibody used for immunoprecipitation recognizes only native non-denatured cytochrome c.
We found that the NOS inhibitor L-NMA inhibited both cytochrome c nitrosylation and Fas-induced apoptosis in CEM cells. However, we and others (29, 54) have previously found that L-NMA stimulates Fas-induced apoptosis in Jurkat cells. This cell line variability may be due in part to the multiple pro apoptotic and anti-apoptotic targets of NO in cells. The net effect of NO on apoptosis may depend on whether the proapoptotic effects of NO such as cytochrome c nitrosylation outweigh the anti-apoptotic effects such as caspase nitrosylation in a particular cell. In addition, the intracellular targets of NO may vary depending on the type and timing of an apoptotic stimulus, the source of NO, and the redox chemistry within a cell (55). Thus, a complex set of factors will determine the net effect of NO on apoptosis in a given cell type.
Our current finding that cytochrome c and previous finding that caspase zymogens (30) are nitrosylated in mitochondria raise the possibility that mitochondria provide a unique redox environment that facilitates protein nitrosylation (30). Caspase zymogens and cytochrome c are located in the mitochondrial intermembrane space where the relatively acidic pH (56) may promote both the formation and stability of metal-NO and S-NO bonds. In addition, mitochondria have a high concentration of lipid membranes that increase the rate of formation of S-nitrosylating equivalents such as nitrogen dioxide (57, 58). Furthermore, complexes III and IV have been shown to reduce nitrite to NO raising the possibility that mitochondria can generate NO via NOS-independent mechanisms (59, 60). Thus, distinctive NO chemistry within mitochondria may allow this organelle to serve as a key site of intracellular protein nitrosylation. Whether nitrosylation targets cytochrome c for release from mitochondria remains to be investigated.
Finally, our results suggest that protein nitrosylation/denitrosylation
may be a mechanism of signal transduction regulation comparable to
phosphorylation/dephosphorylation as first postulated by Stamler (61).
We have shown previously that caspase-3 is inhibited by
S-nitrosylation in resting cells and then is activated by
denitrosylation during apoptosis (29, 30). We now demonstrate that
concurrent with caspase-3 denitrosylation, cytochrome c is nitrosylated, further enhancing caspase-3 activation. The combined data
suggest that cytochrome c nitrosylation and caspase-3
denitrosylation act in concert to stimulate apoptosis (Fig.
4). The findings raise the possibility
that coordinated nitrosylation/denitrosylation of proteins is a general
mechanism of signal transduction regulation in cells.
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ACKNOWLEDGEMENTS |
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We thank Qian Miao for excellent technical assistance, Joe Beckman for insightful comments, and Bob Finberg and Doug Golenbock for support.
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FOOTNOTES |
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* The work was funded by an American Cancer Society Research Project Grant.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.
¶ To whom correspondence should be addressed. Tel.: 508-856-7511; Fax: 508-856-7578; E-mail: joan.mannick@umassmed.edu.
Published, JBC Papers in Press, March 19, 2003, DOI 10.1074/jbc.M212459200
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ABBREVIATIONS |
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The abbreviations used are: Bcl, B cell lymphoma/leukemia; NO, nitric oxide; L-NMA, NG-monomethyl-L-arginine; NOS, nitric-oxide synthase.
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