Mechanism of Superoxide Generation by Neuronal Nitric-oxide
Synthase*
Sovitj
Pou,
Lori
Keaton,
Wanida
Surichamorn, and
Gerald M.
Rosen
From the Department of Pharmaceutical Sciences, University of
Maryland School of Pharmacy, Baltimore, Maryland 21201
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ABSTRACT |
Neuronal nitric-oxide synthase (NOS I) in the
absence of L-arginine has previously been shown to
generate superoxide (O
2) (Pou, S., Pou, W. S., Bredt, D. S.,
Snyder, S. H., and Rosen, G. M. (1992) J. Biol.
Chem. 267, 24173-24176). In the presence of
L-arginine, NOS I produces nitric oxide (NO·). Yet
the competition between O2 and L-arginine for
electrons, and by implication formation of O
2, has until
recently remained undefined. Herein, we investigated this relationship,
observing O
2 generation even at saturating levels of
L-arginine. Of interest was the finding that the frequently
used NOS inhibitor NG-monomethyl
L-arginine enhanced O
2 production in the presence of L-arginine because this antagonist attenuated NO·
formation. Whereas diphenyliodonium chloride inhibited O
2,
blockers of heme such as NaCN, 1-phenylimidazole, and imidazole
likewise prevented the formation of O
2 at concentrations that
inhibited NO· formation from L-arginine. Taken
together these data demonstrate that NOS I generates O
2 and
the formation of this free radical occurs at the heme domain.
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INTRODUCTION |
At a time before the physiologic properties of endothelium-derived
relaxation factor were associated with nitric oxide
(NO·)1 (1-3), it was
found that this free radical activated soluble guanylate cyclase from
crude homogenates of brain tissue (4). The significance of this
observation would, surprisingly, remain dormant for nearly a decade,
even though it was known that L-arginine was the endogenous
activator of this enzyme (5). With the purification and
characterization of a unique monooxygenase, NOS I, capable of oxidizing
L-arginine to L-citrulline, and NO· (6,
7), a new class of small molecules (8) acting as transient second
messengers in the brain was discovered (9). The versatility of this
free radical in controlling a myriad of brain functions will
undoubtedly result in new and provocative findings. Of special interest
will be research that can distinguish NO· from NOS I-secreting
neurons versus NO· from NOS III-containing
endothelial cells and NO· from NOS II-stimulated microglial
cells. Until the recent development of specific antagonists for each of
the NOS isozymes (10-12), it has been difficult to address this
question, which is of particular significance when one considers, as
described in this article, that NOS I, NOS II, and NOS III may produce
NO· and O
2 under differing cell conditions.
Nitric-oxide synthase is known to catalyze the production of NO·
from L-arginine (13, 14). In the absence of substrate, we have previously demonstrated that purified NOS I can use O2
as the terminal electron acceptor, generating O
2 (15). These
findings have been confirmed because O
2 has been spin-trapped
in L-arginine-depleted NOS I-transfected human kidney cells
(16). During the course of our earlier studies (15), we noted to our
surprise that purified NOS I appeared to produce O
2 even in
the presence of L-arginine. The current study, therefore,
explores this phenomenon in depth. Herein, we demonstrate that NOS I,
like NOS II (17) and NOS III (18-20), can generate O
2 and
NO· despite saturating levels of substrate. However, unlike NOS
II, the heme of NOS I is the locus for the production of both free radicals. Finally, we discuss the implications of our findings; particularly relevant is the ability of L-NMMA to enhance
NOS I-derived O
2 even in the presence of saturating
concentrations of L-arginine.
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EXPERIMENTAL PROCEDURES |
Materials--
NADPH, calmodulin, L-arginine,
phenylmethylsulfonyl fluoride, diethylenetriaminepentaacetic acid,
ferricytochrome c, xanthine, NG-nitro-L-arginine methyl ester
(L-NAME), NG-monomethyl
L-arginine (L-NMMA), EGTA, HEPES, and
penicillin G-streptomycin solution were purchased from Sigma.
Imidazole, 1-phenylimazole, sodium cyanide, and diphenyliodonium
chloride (DPI) were obtained from Aldrich. Tetrahydrobiopterin was
purchased from Alexis Biochemicals (San Diego, CA). Cation exchange
resin Dowex 50W-X8 hydrogen form resin was obtained from Bio-Rad.
2',5'-ADP-Sepharose was obtained from Pharmacia (Uppsala, Sweden).
L-[14C]Arginine was purchase from ICN
Radiochemicals (Costa Mesa, CA). Dulbecco's modified Eagle's
medium:nutrient mixture F-12 (1:1) and phosphate-buffered saline were
obtained from Life Technologies, Inc. Bovine calf serum was purchased
from Hyclone (Logan, UT). Superoxide dismutase and xanthine oxidase
were obtained from Boehringer Mannheim. The spin trap
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was synthesized
according to the procedure of Bonnett et al. (21).
NOS I Purification--
NOS I-transfected kidney 293 cells were
cultured in Dulbecco's modified Eagle's medium:nutrient mixture F-12
containing 10% fetal calf serum, penicillin G (100 units/ml), and
streptomycin (100 µg/ml). NOS I was purified from these cells by the
method of Bredt and Snyder (6). Briefly, cells were removed from the culture flasks and washed three times with phosphate-buffered saline
via centrifugation. The pellet was resuspended in buffer containing
phenylmethylsulfonyl fluoride (4 mg/ml) and homogenized with a Polytron
(Brinkmann Instruments, model PCU-2 at setting 2 for 10 s). The
remaining mixture was centrifuged at 15,000 rpm for 20 min to separate
unbroken cells, and the supernatant was applied to a
2',5'-ADP-Sepharose affinity column. After washing the column three
times with 0.45 M NaCl and standard buffer, NOS was eluted
with standard buffer containing 10 mM NADPH. Excess NADPH
was removed by washing and concentrating the eluate with CentriCell-30
(Polysciences, Warrington, PA) until the concentration of NADPH was
approximately 1-1.5 mM as assessed spectrophotometrically at 340 nm (
= 6.2 × 103
M
1 cm
1). Protein concentration
was determined by the Bradford method (22) using bovine serum albumin
as a standard.
Estimation of NOS I Activity--
NOS I activity was assayed by
measuring the formation of L-[14C]citrulline
from L-[14C]arginine as described previously
(23). Briefly, purified NOS I was added into a reaction mixture
containing L-[14C]arginine (0.6 µCi/ml),
L-arginine (1 mM), NADPH (1 mM),
calmodulin (100 units/ml; 23.6 µg/ml), free calcium ions
(CaCl2, 1 µM; calculated as described in Ref.
24), and standard phosphate buffer at pH 7.4 (final volume = 0.150 ml). After incubating at room temperature for exactly 10 min, the
reaction was terminated by adding HEPES (20 mM, 2 ml)
containing EDTA (2 mM, pH 5.5).
L-[14C]Citrulline was separated by passage
through an 0.5-ml column of Dowex-50-X8 cation exchange, and
radioactivity was counted using a Beckman
counter.
Spin Trapping and EPR Spectroscopy--
Spin trapping
experiments with purified NOS I were conducted by mixing all the
components described in the figure legends to a final volume of 0.25 ml. The experiment was initiated by adding freshly purified NOS I. Reaction mixtures were then transferred to a flat quartz cell and
fitted into the cavity of the EPR spectrometer (Varian Associates,
model E-109), and spectra were recorded at room temperature 1 min after
addition of the enzyme. Instrumentation settings are presented in the
figure legends.
Superoxide Detection--
The assessment of the effect of
L-NAME on the ability of xanthine/xanthine oxidase to
produce O
2 was evaluated as described previously (25).
Briefly, xanthine oxidase was added to a solution containing xanthine
(400 µM) and ferricytochrome c (80 µM) such that the rate of O
2 formation, measured
as the SOD-inhibitable reduction of ferricytochrome c at 550 nm (26) was 1 µM/min. The effect of various
concentrations of L-NAME on the rate of O
2
generation was estimated.
NADPH Oxidation--
The rate of NADPH oxidation by NOS I was
determined spectrophotometrically at 340 nm (
= 6.2 × 103 M
1 cm
1).
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RESULTS AND DISCUSSION |
Before determining the ability of NOS I to generate O
2,
our initial series of studies were devoted to determining the
Km and the Vmax for NOS I,
because these constants will help dictate future experimental designs.
Based on these studies, the Km and the
Vmax were found to be 3.47 µM and
216 nmol min
1 mg
1, respectively, well
within the ranges (Km of 2-4.3 µM and
Vmax of 74-3400 nmol min
1
mg
1) reported by others (27).
In the absence of L-arginine, NOS I has previously been
shown to generate O
2 (15, 16), whereas at high concentrations of L-arginine (1 mM), complete inhibition of
O
2 production had been noted (15). We therefore examined the
effect that intermediate concentrations of L-arginine would
have on the ability of NOS I to generate O
2. As shown in Fig.
1, L-arginine, in a
dose-dependent manner, blocked O
2 secretion from
NOS I, exhibiting an EC50 = 5 µM. Inhibition
of NOS I-generated O
2 at 100 µM
L-arginine was nearly complete. Under these conditions, the
rate of NADPH oxidation decreased as L-arginine
concentration increased from 1.96 µmol/min/mg protein in the absence
of substrate to 1.53 µmol/min/mg protein at 100 µM
L-arginine. Thereafter, the rate of NADPH consumption remained constant. This result suggests that for NOS I the transfer of
electrons from NADPH to O2 in the absence of substrate is
faster than the transfer of electrons to catalyze the formation of
NO· and L-citrulline from L-arginine. In
contrast to NOS I, O
2 generation by purified NOS II was
unaffected by 100 µM L-arginine. In fact, it
was still possible to detect this free radical even in the presence of
1 mM L-arginine (17). Similar findings have
been reported for NOS III (18, 19). These are surprising observations, considering the fact that the Km for NOS II varies
between 2.8 to 32 µM, whereas for NOS III, the
Km has been reported to be 2.9 µM
(27). These data nevertheless point to a substantial difference between
the three isozymes of NOS with respect to O
2 secretion. For
NOS II, it was found that O
2 was generated by electron leakage
from the flavin domain (17), based on the finding that 100 µM NaCN did not significantly inhibit O
2
generation as estimated by spin trapping experiments (17). In contrast, either 100 µM NaCN (19) or 1 mM NaCN (18)
blocked O
2 secretion from NOS III. These data suggest that the
heme domain of NOS III is the site of O
2 production (18,
19).

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Fig. 1.
Effect of L-arginine on
O 2 production by purified NOS I as assessed by spin
trapping/EPR spectroscopy. Data are presented as percentages of
control levels in the absence of L-arginine. A typical
reaction mixture contained DMPO (100 mM), free calcium ion
(1 µM), calmodulin (100 units/ml; 23.6 µg/ml), NADPH
(124 µM), and NOS (6.8 µg/ml). Each data point
represents a specific experiment (n = 3) on the same
purified preparation of NOS I.
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The initial step in the generation of NO· by NOS is the
transport of electrons from NADPH to the oxidized flavin, FAD,
resulting in FADH2, after abstraction of a proton from the
surrounding milieu. Disproportionation between the flavins leads to
FADH·/FMNH·. The electron donation from FMNH· to
Fe3+ gives the reduced heme, Fe2+ and
FADH·/FMN
FAD/FMNH· (28). Binding of O2
in the sixth ligand position would give the hypothetical intermediate
[Fe2+-O2] (29). In the absence of
L-arginine, O2 accepts an electron from NOS,
generating O
2 (15, 17-19). When L-arginine is
present, however, there is a binding of the guanidino nitrogen in an
ordered position near the heme (30), which allows the oxidation of
L-arginine to proceed. Based on a Km of
3.47 µM for the NOS I oxidation of L-arginine
and on data presented in Fig. 1, NO· and O
2 are both
generated. The rate of each free radical produced, however, cannot
accurately be estimated by spin trapping. Fig. 1 indicates that NOS I
at L-arginine concentration around the Km is capable of producing O
2 at about 60%
of the rate of that generated in the absence of L-arginine.
For NOS II, electron transport from FMNH· to Fe3+
appears not to be so tightly coupled, because some leakage from the
flavin domain results in the formation of O
2, even in the presence of 1 mM L-arginine (17). For NOS I,
the site of O
2 generation has not yet been defined. We
suggest, however, that during the oxidation of L-arginine
to L-citrulline and NO·, direct competition with
O2 results in O
2 formation. At a fixed concentration of O2, the ratio of NO· and
O
2 is dependent, therefore, on the concentration of
L-arginine.
To explore the mechanism of O
2 generation by NOS I, it is
important to determine how electrons are transferred through the enzyme. Thus, we undertook a series of inhibition experiments exploring
the effects of two well known inhibitors of NOS, L-NAME and
L-NMMA, on O
2 production. Before conducting these
experiments, we estimated the capacity of these NOS inhibitors to block
L-citrulline formation and, by implication, the generation
of NO·. The dose-response curves for
L-[14C]citrulline formation from
L-[14C]arginine by NOS I in the presence of
L-NAME and L-NMMA are shown in Fig.
2. L-NMMA was found to be
slightly more potent (EC50 = 5 µM) than
L-NAME (EC50 = 10 µM). For NOS
III, L-NAME has been reported to be a more effective
inhibitor of this isozyme than L-NMMA (31). With these data
in mind, we then defined the ability of L-NAME and
L-NMMA to prevent O
2 production by NOS I. Fig. 3 shows the effect of increased
concentrations of L-NAME on the ability of NOS I to
generate O
2. Similar to L-arginine,
L-NAME antagonized the spin trapping of this free radical
by DMPO in a dose-dependent manner, with an
EC50 = 40 µM. In contrast, L-NMMA did not appreciably depress the formation of O
2 by NOS I even at concentrations as high as 10 mM (Fig. 3). Consistent
with these findings, the rate of NADPH oxidation by NOS I was inhibited
by >70% as the concentration of L-NAME reached 100 µM, whereas the rate of NADPH oxidation remained constant
at about 50% of control with increasing concentration of
L-NMMA up to 10 mM (Fig.
4). These results suggested that
L-NAME, but not L-NMMA, inhibited the formation
of O
2 by impeding the electron transport to O2. To
confirm this hypothesis, the effect of L-NAME (1 mM) on the xanthine/xanthine oxidase production of
O
2, as measured by the SOD-inhibitable reduction of cytochrome
c, was assessed. Within experimental error, this rate was
unchanged. These data indicate that L-NAME did not scavenge
O
2 but rather that L-NAME acted specifically on
NOS I, inhibiting generation of this free radical. Because
L-NMMA is a potent antagonist of NOS-generated NO·,
we explored whether L-NMMA could block the ability of
L-arginine to inhibit NOS I production of O
2.
These findings are presented in Fig. 5.
As expected, L-arginine (100 µM) almost
completely inhibited the NOS I formation of O
2 (Fig.
5B), which confirmed earlier studies (15, 32). Surprisingly,
L-NMMA, in a dose-dependent manner, reversed
the inhibitory properties of L-arginine (Fig. 5,
C-E), almost reaching control values, in the absence of
L-arginine, at 1 mM (Fig. 5, E and
F). Our findings support the theory that L-NAME
antagonizes the transfer of electrons to either L-arginine or O2, whereas L-NMMA prevents the oxidation of
L-arginine by competing for the same binding site on the
enzyme (33). Although NO· production is inhibited by the
presence of L-NMMA, NOS I still has the capacity to
transfer electrons from NADPH to O2 (Fig. 4 and 5).

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Fig. 2.
Effect of L-NMMA ( ) and
L-NAME ( ) on the generation of L-citrulline
(and by implication on NO·) by purified NOS I. Data are
presented as percentages of control levels in the absence of
L-arginine, as described under "Experimental
Procedures." Each data point represents a typical experiment
(n = 3) on the same purified preparation of NOS
I.
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Fig. 3.
Effect of L-NMMA ( ) and
L-NAME ( ) on O 2 generation by purified NOS I as
assessed by spin trapping/EPR spectroscopy. Data are presented as
percentages of control levels in the absence of either
L-NMMA or L-NAME. Reaction mixtures contained
DMPO (100 mM), free calcium ion (1 µM),
calmodulin (100 units/ml; 23.6 µg/ml), NADPH (124 µM),
and NOS (14.4 µg/ml for L-NMMA; 15.8 µg/ml for
L-NAME). Each data point represents a typical experiment
(n = 3) on the same purified preparation of NOS
I.
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Fig. 4.
Effect of L-NMMA ( ) and
L-NAME ( ) on the rate of NADPH oxidation by purified NOS
I, as described under "Experimental Procedures." For
L-NAME, NOS (4.53 µg/ml) was added into a quartz cuvette
containing free calcium ion (1 µM), calmodulin (100 units/ml; 23.6 µg/ml), and NADPH (124 µM). The control
rate of NADPH oxidation by NOS was 1.27 µmol/mg/min. For
L-NMMA, NOS (10.55 µg/ml) was added into a quartz cuvette
containing free calcium ion (1 µM), calmodulin (100 units/ml), and NADPH (124 µM). The control rate of NADPH
oxidation by NOS was 1.64 µmol/mg/min. Each data point for
L-NAME and L-NMMA represents a typical
experiment (n = 3) on the same purified preparation of
NOS I.
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Fig. 5.
Effect of L-NMMA on O 2
generation by purified NOS I in the presence of L-arginine
as assessed by spin trapping/EPR spectroscopy. Typical EPR spectra
corresponding to DMPO-OOH under a variety of experimental conditions
are shown. The mixture in scan A consists of DMPO (100 mM), free calcium ion (1 µM), calmodulin (100 units/ml), NADPH (124 µM), and NOS (14.4 µg/ml). Scans
B-E were recorded under identical conditions to scan
A except for the addition of L-arginine (100 µM), L-arginine (100 µM) + L-NMMA (10 µM), L-arginine (100 µM) + L-NMMA (100 µM), and
L-arginine (100 µM) + L-NMMA (1 mM), respectively. Scan F was recorded under
identical conditions to scan A except for the addition of
L-NMMA (1 mM). Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G,
sweep time was 12.5 G/min, response time was 1 s, and receiver
gain was 10 × 104.
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Next, experiments were designed to further determine the site on NOS I
where O
2 production takes place. Because cytochrome P-450 and
NOS I are members of the same superfamily of enzymes (34), we looked to
cytochrome P-450 to gain insight as to potential loci for NOS I-derived
O
2. There are two sites on cytochrome P-450 and NOS I where
O
2 may be generated: the flavins of the reductase and the iron
of the heme. For cytochrome P-450, the heme domain is the origin of
O
2 formation (35). Would NOS I behave in a similar manner?
First, we needed to demonstrate that our NOS I preparation was capable
of transferring electrons from the flavin site to the heme domain. For
this purpose we investigated the effect of DPI, a compound known to
block the electron flow at the flavin locus (36), on NO· and
O
2 formation by NOS I. As shown in Fig.
6, DPI is a potent inhibitor of
L-citrulline generation by NOS I with an EC50 = 10 µM. Similarly, DPI at 10 µM almost
completely inhibited the spin trapping of NOS I-secreted O
2
(Fig. 7B). In contrast, DPI
(10 µM) did not inhibit the reaction of DMPO with
O
2 generated from FMN/NADPH (Fig.
8D). Even though these data
indicate that the electron flow is through the flavin domain, these
experiments cannot establish whether O
2 is produced solely
at this site or at the heme domain. To further address this query, we
investigated the effect of NaCN and two imidazoles (known to inhibit
cytochrome P-450 and NOS (37, 38) by blocking the heme site) on the
generation of this free radical. First, however, we had to demonstrate
that NaCN, imidazole, and 1-phenylimidazole inhibited the metabolism of
L-arginine to L-citrulline by NOS I. Fig. 6
depicts the dose-response curves for
L-[14C]citrulline formation from
L-[14C]arginine by NOS I in the presence of
NaCN, imidazole, and 1-phenylimidazole. NaCN was the least potent of
these inhibitors with an EC50 = 10 mM, whereas
imidazole and 1-phenylimidazole were considerably more potent with
values for EC50 = 20 µM and 80 µM, respectively. Once completed, we were confident that
if similar inhibitory properties toward O
2 generation were
revealed, then the heme was most likely the site of this free radical
formation. At 1 mM NaCN, there was a minimal decrease in
the peak height of DMPO-OOH (Fig. 7C), whereas at 10 mM NaCN inhibition of O
2 was nearly complete (Fig.
7D). In an independent series of experiments, NaCN at 10 mM was found to inhibit (by 50%) the spin trapping of
O
2 from the model O
2 generating system consisting of
FMN/NADPH (Fig. 8E). Because NaCN is known to react with
DMPO (21), thereby decreasing the effective concentration of DMPO
available to react with O
2, the findings in Fig. 8E
can only suggest that the heme domain in NOS I was the site of
O
2 production. Alternative antagonists for NOS were sought.
Recently, imidazoles have been reported to block the heme site of NOS
(38). Thus, we investigated the ability of imidazole and
1-phenylimidazole to attenuate O
2 formation by NOS I. As shown
in Fig. 7, 1-phenylimidazole (10 mM) and imidazole (10 mM) inhibited the formation of DMPO-OOH by almost 50 (Fig.
7E) and 80% (Fig. 7F), respectively, whereas,
unlike NaCN, they had no effect on the spin trapping of this free
radical from the O
2-generating system of FMN/NADPH (Fig. 8,
B and C).

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Fig. 6.
A dose-response curve measuring the effect of
1-phenylimidazole ( ), imidazole ( ), DPI ( ), and NaCN ( )
on the generation of L-citrulline (and by implication on
NO·) by purified NOS I. Each data point represents a
typical experiment (n = 3) on the same purified
preparation of NOS I.
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Fig. 7.
Effect of NOS inhibitors on O 2
generation by purified NOS I. Scan A was obtained in
the absence of inhibitors. Scans B-F were obtained in the
presence of DPI (10 µM), NaCN (1 mM), NaCN
(10 mM), 1-phenylimidazole (10 mM), and
imidazole (10 mM), respectively. Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G,
sweep time was 12.5 G/min, response time was 1 s, and receiver
gain was 6.3 × 104.
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Fig. 8.
Effect of NOS inhibitors on O 2
generation by FMN (0.1 mM)/NADPH (1 mM).
Scan A was obtained in the absence of inhibitors. Scans
B-E were obtained in the presence of imidazole (10 mM), 1-phe nylimidazole (10 mM), DPI (10 µM), and
NaCN (10 mM), respectively. Microwave power was 20 megawatts, modulation frequency was 100 kHz with an amplitude of 1 G,
sweep time was 12.5 G/min, response time was 1 s, and receiver
gain was 5 × 104.
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Data gathered from inhibitory experiments with the antagonists
1-phenylimidazole, imidazole, and NaCN support the heme domain as the
site of O
2 production by NOS I, similar to the findings for
cytochrome P-450 (35) (Fig. 9). For NOS
II, it appears that the reductase domain is not as tightly coupled to
the heme domain as seen in NOS I and cytochrome P-450. This weak
coupling allows some electron leakage to O2, generating
O
2, even though sufficient electron flow to the heme permits a
high flux of NO· from the oxidation of
L-arginine.

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Fig. 9.
A proposed model that illustrates O 2
generation from NOS I in the absence of L-arginine.
Initial electron flow is from NADPH to FAD, resulting in
FADH2. After disproportionation with FMN, two semiquinone
free radicals, FADH·/FMNH·, allow electron transport to
the heme iron, giving Fe2+. In the absence of
L-arginine, O2 is the preferred substrate,
resulting in O 2 formation. As there still remains one electron
on FAD/FMNH·, the iron heme is again reduced, with subsequent
transfer to O2. Thus, in the absence of
L-arginine, for every mol of NADPH oxidized, there are 2 mol of O 2 generated.
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Finally, we investigated the role that BH4 might play in
regulating production of O
2 by NOS I in the absence of
L-arginine. Tetrahydrobiopterin, at 10 and 100 µM, inhibited O
2 secretion by NOS I by
>90%. As shown in Fig. 10,
B and C, the EPR spectra are characteristic of
DMPO-OH and to a lesser extent of DMPO-OOH. The source of DMPO-OH
appears to be BH4. At 1 mM BH4
(Fig. 10D), we were still able to observe some DMPO-OOH,
even though DMPO-OH dominated the EPR scan. At 10 mM
BH4, DMPO-OH (Fig. 10E) was the only
spin-trapped adduct recorded. These data suggest that at low
concentrations (1-100 µM), the primary effect of
BH4 is to inhibit NOS I production of O
2. However,
at higher concentrations, in addition to the inhibition of NOS
I-secreted O
2, BH4 generates O
2 through
autoxidation. The resultant H2O2, either
through metal ion catalysis or direct oxidation, produced DMPO-OH (39,
40).

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Fig. 10.
Effect of BH4 on O 2
generation from NOS I in the absence of L-arginine.
Scan A was obtained in the absence of BH4. Scans
B-E were obtained in the presence of BH4 with
increasing concentrations from 10 µM, 100 µM, 1 mM, and 10 mM,
respectively. Microwave power was 20 megawatts, modulation frequency
was 100 kHz with an amplitude of 1 G, sweep time was 12.5 G/min,
response time was 1 s, and receiver gain was 8 × 104 for scans A-D and 2 × 104
for scan E.
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Several important observations are readily apparent from our studies.
First, we were able to spin trap O
2 even in the presence of
saturating levels of L-arginine. This is remarkable,
considering that O
2 and NO· combine at near diffusion
controlled rates, producing ONOO
(41, 42), whereas
O
2 reacts with DMPO at only 12 M
1
sec
1 (43). The ability to spin trap O
2 under
these experimental conditions may result from the fact that O
2
and NO· are generated sequentially at the heme iron site. After
O
2 is produced, NOS I must cycle twice before NO· is
secreted. This is a sufficiently long time to allow O
2 to diffuse from the enzyme to the surrounding milieu where this free radical can react with the high concentration of DMPO included in the
reaction mixture to give the observed spin-trapped adduct, DMPO-OOH.
Under physiological conditions at which NOS I generates both
O
2 and NO·, SOD regulates cellular flux of O
2,
thereby preventing, or at least drastically limiting,
ONOO
generation (44).
Second, despite the above findings, there are pathologic states, such
as ischemia/reperfusion injury, that might promote the formation of
ONOO
(45). Therefore, it is surprising that the fate of
this peroxide still remains in doubt (44, 46). At the low
L-arginine concentrations at which the steady-state flux of
O
2 would exceed NO·, in addition to ONOO
,
H2O2 would arise from the dismutation of
O
2. Subsequent formation of HO·, either through the
metal ion catalyzed Haber-Weiss reaction (47) or from decomposition of
ONOO
(48-52), at sensitive cellular sites may result in
cytotoxicity. Thus, under different experimental conditions, a variety
of oxidants can be produced with an impact on cell function that is
significant or in some cases remains to be defined.
Third, it should be noted that the reductase domain of NOS is, like
cytochrome P-450 reductase, susceptible to uncoupling, shunting
electrons away from the heme toward a xenobiotic. One recent example
illustrates this point (53). It was discovered that
o-quinones can promote O
2 production by uncoupling
NOS I at its reductase domain, yielding a semiquinone free radical. In
the presence of O2, O
2 was generated along with
the parent o-quinone.
In agreement with the result obtained with NOS III (18-20),
BH4, in a dose-dependent manner, was found to
inhibit the generation of O
2 by NOS I. Interestingly, unlike
NOS III, L-arginine, independent of added BH4,
inhibited O
2 production by NOS I, which was reversed by
L-NMMA (Fig. 5). This suggests that in the case of NOS I,
BH4 is not the sole pathway for controlling O
2
formation but that the competition between L-arginine and
L-NMMA is an important factor in determining the generation
of O
2.
How does BH4 regulate the ratio of NO· and
O
2? The answer, of course, is not simple and depends, to a
large extent, on the isozyme of NOS. For instance, one would predict,
based on the findings in Ref. 18, that for an activation of a NOS
III-containing cell in which L-arginine and BH4
are at normal levels, high fluxes of O
2 and NO· would
result. The secretion of NO· to the surrounding milieu would be
tied to the ability of SOD to scavenge O
2. In contrast,
activation of a NOS I-containing cell, under the conditions described
above, would result primarily in the formation of NO·. Thus,
under cellular conditions that would impact the availability of either
L-arginine or BH4, variable amounts of
NO· and O
2 would be generated. Evidence in support of
this thesis comes from studies with NOS I-containing transfected kidney
293 cells (16). Here, when these cells were placed in a complete medium, NO·, but not O
2, was spin trapped (16).
However, when the cells were cultured in
L-arginine-depleted medium for 24 h, it was possible to spin trap O
2 at the expense of NO· (16).
Although the implications of our findings have yet to be fully
realized, two recent publications may shed some light on the importance
of our observations. First, during anoxia/reoxygenation of
cardiomyocytes, generation of O
2 was found to be markedly enhanced when L-NMMA was included (54), which supports the
data presented in Fig. 5. Second, elevated levels of MnSOD were an essential element of viable NOS I containing neurons exposed to N-methyl-D-aspartate (NMDA) (55). Even though
the source of O
2 in these studies (54, 55) was not identified,
NOS must certainly be considered a possible contributor to the origin
of this free radical.
 |
ACKNOWLEDGEMENT |
We acknowledge the technical assistance of
Guan-Liang Cao.
 |
FOOTNOTES |
*
This work was supported in part by Grants AG-14829 and
CA-69538 from the National Institutes of Health.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: Dept. of
Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 West Lombard St., Baltimore, MD 21201. Tel.: 410-706-0514; Fax: 410-706-8184; E-mail: grosen{at}umaryland.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
NO·, nitric
oxide;
O
2, superoxide;
H2O2, hydrogen
peroxide;
NOS I, neuronal nitric-oxide synthase;
NOS II, inducible
nitric-oxide synthase;
NOS III, endothelial nitric-oxide synthase;
DMPO, 5,5-dimethyl-1-pyrroline-N-oxide;
EPR, electron
paramagnetic resonance;
L-NAME, NG-nitro-L-arginine methyl ester;
L-NMMA, NG-monomethyl
L-arginine;
DMPO-OOH, 2,2,-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl;
DPI, diphenyliodonium
chloride;
ONOO
, peroxynitrite;
SOD, superoxide dismutase;
BH4, tetrahydrobiopterin.
 |
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