THEJOURNAL OF BIOLOGICAL CHEMISTRY

Communication Vol. 267, No. 34, Issue of December 5, pp. 24173-24176,1992

Printed in U.S.A.

Generation of Superoxide by brain (3), lung (E),adrenal gland (13), and platelets (14) is a
constitutive enzyme, which requires calcium (11) and calmod-
Purified Brain Nitric Oxide ulin (3) as cofactors. NO participates in signal transduction
Synthase* mechanisms by activating guanylate cyclase to regulate vas-
cular muscle tone, platelet activation(14), neurotransmission
(Received for publication, July 27, 1992) (3,4), and other functions(15, 16). Insight into the structure
Sovitj PouSg, Wanida S. POUS,David S. BredtV, and function of NOS comes from purification and molecular
Solomon H. SnyderV, and Gerald M. RosenS 11 cloning of both the brain and the inducible enzyme (17-20).
From the $Department of Pharmacology and Toxicology, Both enzymes are flavoproteins containing bothflavins FAD
University of Maryland School of Pharmacy, Baltimore, and FMN and an NADPH binding site (17-20). Both NOS
Maryland 21201, the VDepartments of Neuroscience, amino acid sequences displayclose homology with cytochrome
Pharmacology and Molecular Sciences, and Psychiatry and
Behavioral Sciences, Johns Hopkins Medical Institutions,
P-450 reductase, which also contains FAD, FMN, and a
Baltimore, Maryland 21205, and the 11 Veterans recognition site for NADPH. In cytochrome P-450 reductase,
Administration Medical Center, the twoflavinsserve as an electron transport chain from
Baltimore, Maryland 21218 NADPH totheterminal oxidase,cytochrome P-450(21).
More recently, Mayer et al. (22, 23) found that porcine brain
Brain nitric oxide synthase (NOS), which utilizes NOScontainsnon-hemeironand(6R)-tetrahydro-~-biop-
NADPH and calcium/calmodulin as cofactors for me- terin, in addition toFAD and FMN, and generated hydrogen
tabolizing L-arginine to nitric oxide (NO) and L-citrul- peroxide at suboptimal concentrations of L-arginine. In the
line, contains recognition sites for the flavins FAD and present study we show that purified NOS produces 0, in a
FMN. Using a spin-trapping technique combined with NADPH and calcium/calmodulin-dependent manner.
electron spin resonance spectroscopy, we report that
brain NOS generates superoxide 0;in a calcium/cal- MATERIALS ANDMETHODS
modulin-dependent manner. The ‘specific inhibitors* Reagents-Diethylenetriaminepentaacetic acid (DTPA),EGTA,
of NOS, NG-monomethyl L-arginine (L-NMMA),and ferricytocbrome c (type VI), NADPH, calmodulin, p-nitro-L-argi-
NG-nitro-L-argininemethyl ester (L-NAME), have dif-nine methyl ester (L-NAME), xanthine, phenylmethylsulfonyl fluo-
ferenteffects on 0; generation. For L-NMMA, 0; ride, andpenicillin/streptomycinsolution were purchased from
production is unaffected, while for L-NAME,inhibition Sigma. p-Monomethyl L-arginine (L-NMMA) was from Research
of this free radical is concentration-dependent. Biochemicals Inc. (Natick, MA). 2’,5’ADP-Sepharose was from Phar-
macia Fine Chemicals (Uppsala, Sweden). [14C]~-Arginine was from
ICN Radiochemicals Inc. (Costa Mesa, CA). Superoxide dismutase,
catalase, and xanthine oxidase were obtained from Boehringer Mann-
Nitric oxide (NO) possessesseveral physiologic roles in heim. Dulbecco’s modified Eagle’s medium:nutrientmixture F-12
mammalian systems, mediating endothelial-derived relaxa- (1:l)was from GIBCO. Bovinecalf serum was from HyClone (Logan,
UT). The standard buffer system used was 50 mM potassium phos-
tion of vascularsmooth muscle (1, 2), actingassynaptic phate buffer, p H 7.4, containing DTPA (1 mM) and EGTA (1 mM).
neuronal messenger (3,4) and accountingfor cytotoxicactions Free calcium concentration was calculated as previously described
of macrophages ( 5 ) .At least two distinct formsof nitric oxide (24). The spin trap 5,5-dimethyl-l-pyrroline1-oxide (DMPO), syn-
synthase (NOS),’ which catalyzes the formation of NO and thesized as described (25), was purified by distillation.
L-citrulline fromL-arginine, have beendescribed. In activated NOS Purification-NOS-transfected kidney 293 cells were grown
macrophages, Kupffer cells (6), hepatocytes (7), and neutro- in Dulbecco’s modified Eagle’s medium:nutrient mixture F-12 con-
phils (8),NOS is calcium-independent, requires (6R)-tetra- taining 10% fetal calf serum and penicillin (100 units/ml), strepto-
mycin(100Fg/ml). Enzyme purificationfrom these cells was as
hydro-L-biopterin, and is an immunologically inducible en- described previously (26) withminor modifications. Briefly, cells were
zyme (9, 10). NOS found in the vascular endothelium ( l l ) , removed fromthe flasks andwashed three timeswith standard buffer.
The pellet was resuspended in buffer containing phenylmethylsulfo-
* This work was supported in partby National Institutes of Health nyl fluoride (4 pg/ml) and homogenized with a Polytron homogenizer
Grant HL-33550, the Veterans Administration Research Service, the (Brinkmann Instruments). After centrifugation, the supernatantwas
Council for Tobacco Research U.S.A., the Maryland Industrial Part- applied toa 2’,5’-ADP-Sepharose affinity column. After washing the
nerships, United States Public Health Services Grant MH-18501, column with 0.45 M NaCl, NOS was elutedwithstandard buffer
Contract DA-271-90-7408, Research Scientist Award DA-00074 (to containing 10 mM NADPH. The excess of NADPH was reduced by
S. H. S.), Training Grant GN-07309 (to D. S. B.), and a gift from washing and concentrating the eluate with Centricell-30 (Polysci-
Bristol-Myers Squibb. The costs of publication of this article were ences, Inc.) until the concentrationof NADPH was about 1-1.5 mM.
defrayed in part by the payment of page charges. This article must For eachexperiment enzymewasfreshly prepared. An identical
therefore be hereby marked “aduertisement” in accordance with 18 protein purification procedure was also employed with untransfected
U.S.C. Section 1734 solely to indicate this fact. 293 cells. NOSactivity was assayedby the conversion of [ I 4 C ] ~ -
§ T o whom correspondence should be addressed: Dept. of Phar- arginineto[14C]~-citrullineas previouslydescribed (17).Protein
macology and Toxicology, University of Maryland School of Phar- concentration was determined by the Bradford method with bovine
macy, 20 N. Pine St., Baltimore, MD21201. Tel.: 410-706-7358; Fax: serum albumin as a standard (27).
410-706-7184. SpinTrapping-Spin-trappingexperiments were performed by
The abbreviations used are: NOS, nitric oxide synthase; SOD, mixing all the componentsdescribed in the figure legends to a final
superoxide dismutase; OF, superoxide; DMPO, 5,5-dimethyl-l-pyr- volume of 0.25 ml. The reaction was initiated by adding NOS. Spin
roline 1-oxide; DMPO-OOH, 2,2-dimethyl-5-bydroperoxy-l-pyrroli-trapping in thepresence of NO was performed by first allowing NOS
dinyloxyl; DMPO-OH, 2,2-dimethyl-5-hydroxy-l-pyrrolidinyloxy1; to generate superoxide for 1.5 min, after which DMPO and 0.2 ml of
DTPA, diethylenetriaminepentaacetic acid; L-NAME,P-nitro-L- NO gas were added to the reactionmixture. Since the additionof 0.2
arginine methyl ester; L-NMMA, Nc”monomethy1 L-arginine; ESR, ml of NOresultedin adecrease in pH from 7.4 to 6.8, control
electron spin resonance; EDRF, endothelial-derived relaxation factor.experiments were performed by adding the same amount of NO to

24173
24174 Superoxide
Generation by Brain
Nitric Oxide Synthase
the buffer and by allowing it to stand at room temperature for 10-15
min prior to the addition of NOS and DMPO. NO was introduced
into the reaction mixture by a gas-tight syringe. At standard temper-
ature and pressure, 0.2 ml of NO equals 4.4 mmol (28). Reaction
mixtures were then transferred to a flat ESR quartz cell, fitted into
the cavity of the spectrometer (Varian Associates E-109), and the
spectra were recorded 1min after the addition of the enzyme at 25 "C.
Superoxide Detection-The rate of 0, formation from NOS was
determined as the superoxide dismutase (SOD)-inhibitable reduction
of ferricytochrome c and monitored spectrophotometrically at 550
nm, as previously described (29).
NADPH Oxidation-The rate of NADPH oxidation by NOS was
determinedspectrophotometrically at 340 nm (C = 6.2 X lo3 M"
cm").

RESULTS ANDDISCUSSION
The rationale behind our studies is the close amino acid
sequence homology between cytochrome P-450 reductase and
NOS,thefactbrainNOShasbeenreportedtogenerate
hydrogen peroxide (22, 23), and the fact that cytochrome P -
450 reductase is known to form O, (21). Therefore, using D
NOS purified from astable cell line thatexpresses brain NOS,
we investigated the possible generation of 0, by NOS. Ini-
tially, we attempted to measure 0, flux spectrophotometri-
cally by monitoring the SOD-inhibitable reduction of ferri-
cytochrome c. However, inthepresence of NADPHand
calcium/calmodulin, NOS catalyzed the reduction of cyto-
chrome c, which was not inhibited by the inclusion of SOD
(30 units/ml, data not shown). This finding suggests either
that 0; is not generated by NOS or that the one-electron
reduction of ferricytochrome c by NOS is faster than the
reduction of this iron-containing enzyme by 0;. Therefore,
we chose to use spin trapping combined with ESR spectros-
copy (30)todetermine if 0, isgenerated by NOS. This -F
technique consists of using a nitrone or a nitroso compound
to trap the initial unstablefree radical as a long-lived nitrox- G -
ide, which can beobserved by ESR spectroscopy (31). For our FIG. 1. ESR spectra from purified NOS. ESR spectra were
purpose the spin trap DMPO was used to detect0;. obtained from the aerobicaction of NOS. The mixture in scan A
When DMPO (100 mM) was added to purified NOS (7 gg/ consists of purified NOS (7 pg/ml), NADPH (124 p ~ )free , calcium
ml) containing NADPH (124 g ~ in) the presenceof calcium (1FM) calmodulin (100 units/ml), and DMPO (100 mM). Scans B-c
(1g ~and ) calmodulin (100 units/ml), an ESR spectrum was were recorded under conditions identical to those for scan A, except
recorded in which the predominantspecies was characteristic for the omission of calciumandcalmodulinin scans B and C,
respectively. Scans D-F were recorded under conditions identical to
of the 0, spin-trapped adduct 2,2-dimethyl-5-hydroperoxy- those for scan A except for the addition of NADP+ (200 mM), catalase
1-pyrrolidinyloxyl (DMPO-OOH). A small signal character- (300 units/ml), SOD (60 units/ml) in scans D , E, and F, respectively.
istic of the hydroxyl radical spin-trapped adduct 2,2-dimethyl- Scan G was obtained under conditions identicalto those for scan A
5-hydroxy-1-pyrrolidinyloxyl(DMPO-OH) was also detected except that NOS was replaced by the protein isolated from the original
(Fig. 1A). These ESR signals were observed for at least 20 cellslacking NOS. ThesignalscorrespondingtoDMPO-OHand
min. Since the half-life of DMPO-OOH is about 8 min, this was DMPO-OOH are designated 1 and 2, respectively. Microwave power
20 milliwatts, modulation frequency was 100 kHz with an ampli-
result indicates that under the proper conditions, NOS is tude of 1 G, sweep time was 12.5 G/min, response time was 1 s, and
capable of sustaining the generation of oxygen centered free receiver gain was 8 X IO4.
radicals for a long period of time. In the absence of calcium
or calmodulin and in the presence of a large excessof NADP+
(200 mM), only a small DMPO-OH signalwas observed (Fig. these oxygen centered free radicals,weperformed an identical
1, B-D, respectively), which was inhibited by SOD (60 units/ protein purification procedure on the original cell line, which
ml). ESR spectra corresponding to DMPO-OOH and DMPO-lacks NOS. Using this protein preparation, neither oxygen-
OH were not affected by catalase (300 units/ml) but were centered free radicals (Fig. 1G) nor the conversion of [ l 4 C ] ~ -
completely inhibited by the inclusion of SOD (60 units/ml) arginine to [14C]~-citrulline was detected. These observations
(Fig. 1, E and F ) . The effects of SODandcatalaseare unambiguously demonstrate that NOSis capable of reducing
consistent with 0, as the only oxygen-centered free radical molecular oxygen by one electron to 0;. Heinzel et al. (23)
formed by NOS with the DMPO-OH signal resulting from suggested that brain NOS reduced molecular oxygen by two
the decomposition of DMPO-OOH as previously reported electrons to hydrogen peroxide. However our data suggested
(32). an alternative pathway in which hydrogenperoxide arises
The sameenzyme preparation alsocatalyzed the formation from the dismutation of 0;.
of [14C]~-citrulline from [I4C]~-arginine a calcium/calmod-
in To investigate in greater detail the mechanismof 0; gen-
ulin- and NADPH-dependent manner, indicating that the eration by NOS, we examined the effect of the natural sub-
preparation used in the spin-trapping experiments contains strate L-arginine and two specific inhibitors of NOS, p -
catalyticallyactive NOS. As additionalconfirmationthat monomethyl L-arginine (L-NMMA) and P-nitro-L-arginine
NOS rather than other uncharacterized proteins produced methylester(L-NAME)onthespintrapping of this free
 Superoxide
Generation by Brain
Nitric Oxide Synthase 24175
radical. L-Arginine(1mM, Fig. 2B)inhibited the spin trapping
of 0,. There areseveral possibleexplanations for this finding.
First, since L-arginine did not inhibit the rate of NADPH
oxidation, it may compete effectively with molecular oxygen
for the electrons donated by NADPH. As such theproduction
of 0; would be inhibited, thereby decreasing the ESR spec-
trum for DMPO-OOH.Alternatively, the amount of 0 ; gen-
erated is not decreased; instead, the reactionof NO with 0,
to form peroxynitrite (33) would diminish the concentration
of 0, available to react with DMPO. Finally, since NO is a
free radical,it could react with the
nitroxide moietyof DMPO-
OOH t o give a diamagnetic species, which would not be visible
by ESR spectroscopy. To test this latter hypothesis, we in-
cubated the nitroxide 2,2,6,6-tetramethyl-l-piperidinyloxy(1
PM) with either an NO-generating system, sodium nitroprus-
side at various concentrations (1-10 mM), or NO gas (4.4
mmol). Under all experimental conditions, the ESR spectrum
was not diminished, therefore excluding this possibility. Next,
studies were designed to discriminate between the other two
hypotheses by adding NOgas to the reactionmixture. As
shown in Fig. 3, inclusion of 4.4 mmol of NO gas resulted in
a significant decrease in the ESR signalcorresponding to
DMPO-OOH for the first 5 min. These findings indicated
FIG. 2. Effect of L-arginine and specific inhibitors of NOS that in the presenceof NO, superoxide preferentially reacted
on superoxide formation. The mixture in scan A consists of with NO rather than being spin-trapped by DMPO. Thus,
purified NOS (1.9 pg/ml), NADPH (108 p ~ ) free , calcium (1 p ~ ) , particularly when the endogenous concentration of L-arginine
calmodulin (100 unitslml), and DMPO (100 mM). Scans B-D were is low, the calcium/calmodulin-activated NOS resembles the
recorded under conditions identical to those for scan A except for the
addition of L-arginine (1 mM), L-NAME(1 mM), and L-NMMA (10 plasma membrane-associated NADPH-dependent oxidase of
mM) in scam B , C, and D, respectively. Instrument settings were the neutrophils, which reduce molecularoxygen to 0 ; and hydro-
same as noted in Fig. 1. gen peroxide during microbial challenge.
For L-NAME,like L-arginine, the ESR spectrumwas again
markedly decreased. However,the explanationfor this finding
is different. Addition of L-NAME (1mM, Fig. 2C)to NOS in
the presence of NADPH resulted in an inhibition in the rate
of NADPH oxidation. Therefore, the decrease in the spin
Control trapping of 0, is associated with an inhibition in the forma-
NOgas tion of this free radical.
At concentrations up to 10 mM L-NMMA had minimal
inhibitory effect on 0, detection (Fig. 2 0 ) . Furthermore, the
rate of NADPH oxidation by NOS in the presence of either
L-arginine (1 mM) or L-NMMA (10 mM) was similar. This
finding is consistent with the observation of Heinzel et al.
(23) that L-NAME but not L-NMMA inhibits hydrogen per-
oxide formation from brain NOS. This resultmay also explain
the findings of Thomas et al. (34), who suggested that 0;
may participate in the vascular effects of L-NMMA. They
found that SOD significantly attenuates the vasoconstrictor
effect of L-NMMA.
! . ! . , . , 6. , .8 , . 1 0 ,
00 12 The finding that NOS generates 0, during its enzymatic
cycling has important implications. Peroxynitrite, resulting
Time (min) from the reaction of 0, with NO, both formed by NOS, might
explain the shortpharmacological lifetime of EDRF and the
FIG. 3. Time course of NOS superoxide generation in the ability of SOD to prolong the action of EDRF. Differing
presence of NO. The mixture in the curue with open circles serves
as a control and consists of purified NOS (30 pg/ml), NADPH (100 pharmacological effects of L-NMMA and L-NAME may re-
pM), free calcium (1 p M ) , calmodulin (100 units/ml), DMPO (100 flect differing mechanisms of NOS inhibition. In the case of
mM), and sufficient buffer (0.5 ml, final volume) in which 0.2 ml (4.4 L-NAME, inhibition of NO production concomitant with a
mmol) of NO gas had been added and allowed to stand 15 min open decrease of 0, appears to result from an antagonism of the
to air. The curue with closed circles was identical to the open circle NOS electron transport chain. For L-NMMA, inhibition pre-
curue except that 0.2 ml of NO gas was added at the same time as
DMPO to the reaction mixture.Inbothtracings NOS was first sumably involves a different site than L-NAME, since 0,
allowed to generate superoxide for 1.5 min prior to the addition of flux is unaltered.
DMPO. Height of the first low field peak of DMPO-OOH was
determined at 1-min intervals after the addition of DMPO. Instru- Acknowledgments-We thank Dr. Daniel M. Ziegler for helping us
ment settings were the same as noted in Fig. 1, except the receiver in the enzymology of nitric oxide synthase, Dr. Terry B. Rogers for
gain was 3.2 X lo4.Each time point represents a mean k S.D. of three providing us with a program for calculating the concentration of free
measurements. calcium, and Mike Gentry and Rahel Teferi for assistance.
24176 Superoxide
Generation
Nitric
Brain
by
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