ABSTRACT. The cofactor requirements of macrophage nitric oxide. (NO.) synthase suggest involvement of an. NADPH-dependent flavoprotein. This prompted.
Inhibition
of macrophage
synthase
and
endothelial
by diphenyleneiodonium
DENNIS J. STUEHR,2 JOSE A. GONZALEZ,
OLUFUNMILAYO ROBERTO LEVI,*
cell nitric and
A. FASEHUN,* NYOUN AND CARL F. NATHAN
its analogs
oxide 1
SOO KWON, STEVEN
S. GROSS,*
The Beatrice and Samuel A. Seaver Laboratory, Division of Hematology, Department of Medicine and *Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA
The (NO.)
ABSTRACT
nitric
oxide
cofactor synthase
NADPH-dependent test the effect
iodonium
requirements of macrophage suggest involvement of an
flavoprotein.
of the flavoprotein
This
prompted
inhibitors
us to
diphenylene-
(DPI),
di-2-thienyliodonium (DTI), and iodoon the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and niumdiphenyl
(ID)
partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (1C50’s 50150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP’, or 2’5’-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once N0 synthase was inhibited by DPI,
neither also
NADPH
inhibited
nor FAD could restore its activity. acetylcholine-induced
epinephrine-preconstricted
relaxation
rabbit
aortic
DPI
of nor-
rings (1C50 300
nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO. -generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and en-
dothelial
cells depends
on an NADPH-utilizing
flavopro-
tein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO..Stuehr, D. J.; Fasehun, 0. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.; Nathan, C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEBJ. 5: 98-103; 1991. Kty
Words:
titelium-derived
NITROGEN
L-arginine relaxing
OXIDE
demonstrated
FAD
BIOSYNTHESIS
in
fiavopmlein
.
inteiferon-y
.
endo-
factor
FROM
L-arginine
has
METHODS Materials (6R,S)-5,6,7,8-Tetrahydro-L-biopterin ADP Sepharose, the murine 264.7,
and
recombinant
mouse
(BH4), macrophage interferon-y
NADPH, cell line were
2,5RAW
obtained
from the same sources as previously reported (23). All other reagents were from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise noted. The sulfate salts of DPI, DTI, and ID were a gift from Dr. Andrew Cross (University of Bristol, England), and were stored in powder form at -80#{176}C.Solutions (2-5 mM) were freshly prepared in dimethylsulfoxide. Stock solutions of NADPH, GSH, BH4, and FAD were prepared fresh for each experiment in 40 mM Tris-HCI, pH 8.0. GSH solutions were brought to pH 7.2 with 1 M NaOH before use. None of the additives or inhibitors tested alone
been
(1, 2), endothelial cells (3, 4), neutrophils (5), tumor cells (6), Kupifer cells (7), and hepatocytes (8).The products of this novel enzymatic reaction are L-citrulline and the reactive radical nitric oxide (NO.)3 (9-12). This pathway may participate in regulating
‘From the Minisymposium “Macrophage Biology, III” presented at the joint meeting of the American Society for Biochemistry and Molecular Biology and the American Association of Immunologists, June 4, 1990, New Orleans, Louisiana. Chaired by S. Vogel and T. A. Hamilton.
several
2To whom reprint requests should be addressed, at: Box 57, Cornell University Medical College, 1300 York Ave., New York, NY 10021, USA.
physiological
isolated
which differ in their regulation, cofactor requirements, and inhibitor profiles (21). In macrophages, interferon-’y and bacterial lipopolysaccharide induce a soluble NO. synthase that is L-arginineand NADPH-dependent (17, 22). The partially purified macrophage NO. synthase also requires as cofactors tetrahydrobiopterin (BH4) (23, 24), FAD, and thiol, typically GSH (25). The requirement for FAD and NADPH suggested that macrophage NO. synthase may contain a flavoprotein (25). To investigate this further, we used diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) (Fig. 1), inhibitors of several nucleotide-requiring flavoproteins (26-30). We report here that DPI and its analogs are potent, irreversible inhibitors of NO. synthase in both mouse macrophages and rabbit aortic rings, and that their inhibitory effects are specifically antagonized by NADPH and FAD. These findings strengthen the evidence for an NADPH-utilizing flavoprotein in NO. synthesis and introduce a new class of inhibitors for the study of NO. physiology.
processes
macrophages
including
vascular
tone
(3, 4),
cerebellar signaling (13, 14), platelet adhesion (15), and macrophage anti-tumor and anti-microbial activity (10, 16-18). NO. mediates its effects by activating some enzymes, such as soluble guanylate cyclase (4), and by inhibiting others, such as succinate:ubiquinone oxidoreductase, NADH:ubiquinone oxidoreductase, and cis-aconitase (10, 16, 19, 20). There appear to be at least two forms of NO. synthase,
98
‘Abbreviations: ACh, acetylcholine; BH4, (6R,S)-5,6,7,8-tetrahydrobiopterin; DPI, diphenyleneiodonium; GSH, reduced glutathione; ID, iodoniumdiphenyl; DTI, di-2-thienyliodonium; NO, nitric oxide; N02, nitrite; NO3, nitrate; ADPR, adenosine 5-diphosphoribose; RFLAV, riboflavin; SNP, sodium nitroprusside; RPHPLC, reverse phase high-performance liquid chromatography.
0892-6638/91/0005-0098/sw
.50. © FASEB
ID
NADPH, 2 mM L-arginine, 4 tM BH4, 4 tM FAD, and 5 mM GSH. In some cases, control macrophage cytosol (20 -160 mg) was added to the assay in place of FAD, BH4, and GSH. Reactions were terminated by adding two units of L-lactic dehydrogenase and 5 mol sodium pyruvate, and incubating further for 15 mm. This procedure oxidizes residual NADPH as determined spectrophotometrically, which otherwise interfereswith the colorimetric assay for N02 and NO#{231}. The combined concentration of NO2 and N03 in the incubates was assayed as described previously (22). Culture
DTI
of peritoneal
macrophages
Thioglycolate-elicited peritoneal macrophages were obtained from CD-i mice and plated at 10’ cells per well in 96-well microplates as previously described (16). Macrophage NO. synthase activity was induced by incubating the cells with interferon-’y (500 units/mi) and 1 ig/ml Escherichia coli lipopolysaccharide for 6-12 h. The culture medium was then replaced with 100 sl medium containing various concentrations of DPI, DTI, or ID. after culture for 24 h, the NO2 plus N03 concentration in the medium was measured. The N02 plus NO, concentration in cell-freemedium (5-8 jzM) was subtracted from each value to quantify NO. synthase activity. Treatment
of enzyme
preparations
with
DPI,
DTI, or ID
Activated RAW 264.7 macrophage cytosol (1 mg, 100 sl) or semipurified NO. synthase (50 eg in 100 1d) was mixed with 100 fLi of 40 mM Tris-HC1 buffer, pH 7.2. DPI (10 LM), DTI (50 fLM), or ID (50 &M) were added, and the samples were incubated for 30 mm at 4 or 37#{176}C. Then I ml of ice cold buffer was added, the samples were immediately transferred to Centricon-30 microconcentrators, and their volume was reduced to 50-100 11 at 4#{176}C. Dilution with 1 ml buffer and concentration were repeated three more times, after which the sample volume was brought up to 300 sl. The protein Figure 1. Iodoniumdiphenyl (ID), di-2-thienyliodonium ([ff1), concentration of each sample was then measured by the Bradford method (31) using the Bio-Rad reagent (Richand diphenyleneiodonium (DPI). mond, Calif.), and NO. synthase activity of 20 sl aliquots or in combination interfered with enzymatic removal of was assayed as described above. All values obtained were residual NADPH (see below) or with the assay for nitrite normalized for protein. Controls not receiving DPI were (NO,) and nitrate (NO,-). processed in an identical manner. When semipurified NO. synthase was used, the initial dilution after the DPI incubaPartial purification of NO synthase tion was done with buffer containing 1 mg/mI bovine serum Cytosols of nonactivated (control) and interferon-’y/lipoalbumin as a protein carrier. Where indicated, putative DPIpolysaccharide-activated (induced) RAW 264.7 macrophages blocking agents (NADPH, FAD, and their derivatives)were were prepared as detailed in ref 22. NO. synthase was parpreincubated with NO. synthase preparations for 10 mm at tially purified by affinity chromatography of the induced 37#{176}C before adding DPI and incubating further for 30 mm. macrophage cytosol on 2’ ,5’-ADP Sepharose as described For experiments that tested whether enzyme catalysis (25). alters the kinetics of NO. synthase inhibition by DPI, we required a preparation of NO. synthase free of cofactors and Measurement of NO. synthase activity L-arginine. For this,NO. synthase activityof 1 ml of activated macrophage cytosol was separated from endogenous NO. -generating activitywas quantitated on the basis of the substrate and cofactors by chromatography on Sephadex NADPHand L-arginine-dependent formation of NO2 G-25, concentration to 0.1 ml in a microconcentrator, and plus NO3- in the incubation mixtures. NO2- and NO,- are washing three times with 1 ml buffer before use. the stable, accumulating oxidation products of NO., and their measurement serves as a convenient assay for NO. Aortic rings production. Reactions were carried out at 37#{176}C for 3 h in New Zealand White rabbits (2-3 kg) were lightly anesthe96-well microplates (Corning Glass Works, Corning, N.Y.), as previously described (25). Unless otherwise noted, reactized with CO, and exsanguinated. The thoracic aorta was tions contained either 1-5 &g of semipurified NO. synthase removed and cut into rings 3-4 mm wide. Rings were susor 50 tg of induced, unfractionated macrophage cytosol. pended under a resting tension of 4.0 g in 5-ml glass chamReactions were carrier out in buffer (40 mM Tris-HCI, pH bers containing Krebs-Henseleit solution gassed with 95% 8.0) supplemented with protease inhibitors (22), 2 mM 02 and 5% CO2 at 37#{176}C. Rings were contracted with 1 eM
DPI
IRREVERSIBLE INHIBITION
OF NO SYNTHASE
99
norepinephrine; changes in tension were recorded with an isometric force-displacement transducer (Model FTO3, Grass Instruments, Quincy, Mass.) and displayed on a polygraph (Grass Instruments).
TABLE
1. Macropliage
NO
synthase
is irreversibly
inhibited
Activated
Assayed
with:
DPI,
10
vM
by DPI”
Semipurified synthase
cytosol
N0
nmol nitrite plus
RESULTS
Defined
Inhibition of macrophage DPI, DTI, and ID
NO.
synthase
cofactors
Nonactivated
When present throughout the 3-h assay, DPI, DTI, and ID each inhibited the activity of semipurified macrophage NO. synthase in a concentration-dependent manner, with IC,0’s of 50, 150, and 150 nM, respectively (Fig. 2A). Similar results were obtained when unfractionated cytosol from activated macrophages was used as a source of NO. synthase activity (not shown). DPI, DTI, and ID also inhibited NO. synthesis over a 24-h period by intactactivated mouse macrophages, with IC,0’sof and 30, 80, and 80 nM, respectively (Fig. 2B). Concentrations 3 M were nontoxic to macrophages, as determined by trypan blue dye exclusion. Inhibition of macrophage is irreversible
NO.
synthase
by DPI
+
41.0 ± 0.4 1.7 ± 0.2
25.5 ± 0.1 1.3 ± 0.1
-
32.2
± 0.6 1.0 ± 0.3
22.0 ± 0.3 0.3 ± 0.1
-
by cytosol
nitrate
+
“Activated macrophage cytosol or NO synthase were incubated for 30 mm at 37#{176}C with or without DPI. After removal of unbound inhibitor, NO synthase activity was assayed in the presence of defined cofactors or nonactivated macrophage cytosol as described in Methods. The NO,plus
NO, values experimental
were normalized based on the protein concentration sample at the point of assay, and are the means three incubations.
of each sD of
±
BH4 (4 ftM) (i.e., assay conditions allowing for enzyme catalysis), the time course of irreversible inhibition was unaltered (Fig. 3). Specificity
of inhibition
by DPI
Millimolar concentrations of NADPH can prevent DPI from inhibiting neutrophil NADPH:O, oxidoreductase (26). The Cross and Jones (26) found that DPI irreversibly inhibited upper panel of Fig. 4 shows that irreversible inhibition of the neutrophil flavoprotein NADPH:02 oxidoreductase. Likewise, NO. synthase activity of either a semipurified enmacrophage NO. synthase by 10 sM DPI was substantially zyme preparation or activated macrophage cytosol was irblocked when 5 mM NADPH was present during the reversibly inhibited after a 30-mm incubation with 10 sM 30-mm incubation with DPI. Equivalent protection was DPI at 37#{176}C (Table 1). This was true whether NO. synthase afforded by NADPH, NADP, and the NADP(H) structural activity (after DPI treatment and washing) was assayed fragment 2’,S’-ADP (5 mM each). In contrast, 5 mM NADH was completely incapable of protecting NO. synthase from using defined additives (FAD, GSH, BH4) (25) or cytosol from nonactivated macrophages as a source of these cofactors (22, 23). The onset of irreversibleinhibition was temperature-dependent, being diminished from 97 ± 2% (n = 4) complete to 44 ± 23% (n = 4) complete when the 30-mm DPI treatment was carried out at 4 instead of 37#{176}C (not shown). Similar results were obtained when 50 sM DTI or ID substituted for DPI (not shown). > r’) In the absence of L-arginine and cofactors, irreversible in-4-, L. hibition of NO synthase activity required 10 mm exposure a) to 10 eM DPI at 37#{176}C (Fig. 3). If instead the 30-mm DPI .1 a) treatment was carried out in the presence of L-arginine (2 u,I mM), NADPH (2 mM), FAD (4 ftM), GSH (5 mM), and 0 (‘1
-Co -I-’
>‘-
9
1
(1)0
0
E
OC
z
6
0
z +
0
0
2
0
3
10 Exposure
0
0 -9
-8
-7
-6
Concentration,
-9 log
-8
-7
-6
M
2. Inhibition of semipurified N0#{149} synthase (A) or activated macrophage NO#{149} synthase (B) by DPI (0), IT (is), or ID (S). NO,- + NO, values in controls not receiving inhibitors were 5.1 ± 0.3 nmol (A) and 7.9 ± 0.4 nmol (B). Each point is the mean ± SD of three incubates. Error bars not shown were contained within the symbols.
Figure
100
Vol. 5
January
1991
Figure
3. Time course of irreversible
20
30
to DPI, mm inhibition
by DPI. Desalted,
activated macrophage cytosol (400 g) was preincubated for 10 mm at 37#{176}C in 0.5 ml buffer (0) or buffer containing 2 mM L-arginine, 0.25 mM NADPH, 4 cM BH4, 4 tM FAD, and 5 mM GSH (S). After addition of DPI (10 tiM), 0.1-mi samples were removed at the times indicated and immediately diluted in 4#{176}C buffer containing 500 tM FAD. After washing of unbound DPI from each sample, NO synthase activity was assayed as described in Methods. Values are the mean ± SD of three incubates.
The FASEB Journal
STUEHR ET AL.
DPI inhibition. The lower panel of Fig. 4 shows that either FAD or FMN (0.5 mM each) were also effective in blocking irreversible inhibition by DPI. However, the FAD structural fragments riboflavin (RFLAV) or adenosine 5-diphosphoribose (ADPR) at 0.5 mM did not serve as protectants. If NADPH (5 mM) or FAD (0.5 mM) were added to NO. synthase after its 30-mm incubation with DPI, neither reversed inhibition(not shown). To testwhether FAD or NADPH reacted with DPI in solution, each was incubated with DPI (30 mm at 37#{176}C in 40 mM Tris-HC1 buffer, pH 8.0) and analyzed by reverse phase high-performance liquid chromatography (RPHPLC). The retention times and peak areas for FAD and NADPH were identical to controls not receiving DPI (not shown). In contrast to FAD and NADPH, none of the other assay components (100 LM BH4, 5 mM GSH, or 2 mM L-arginine) was able to protect NO. synthase from inhibition by DPI (not shown). Inhibition of release of endothelium-derived factor in intact vascular rings
relaxing
Like mouse macrophage NO. synthase, NO. synthase activity in lysates of porcine aortic endothelial cells requires NADPH (32). To test whether endothelial cell NO. synthase also resembles the macrophage enzyme in its susceptibility to inhibition by DPI, we tested the abilityof DPI to block acetylcholine (ACh)-induced relaxation of norepinephrineconstrictedrabbit aorticrings,an action mediated by release
AddItIve DPI I
I
I
I
I
I
I
I
I
I
+ NADPH + NADP
+
75’ADP + NADH +
-I
AdditIve DPI I
FAD
C 0
0
60
a
20
E 40 E
40
0 V
0
60.
E 0
0 0
x
20
C
80
0 .0
C
0 0.01
1
0.1
10
0.01
DPI, #{216}4
0.1
1
10
Acetylcholine,
Figure 5. Leftpanel)Concentration-responserelationship forinhibition by DPI of the maximum relaxation elicited by 1 M acetylcholine (ACh) in rabbit aortic rings. ACh-induced relaxation of norepinephrine (1 LM)-constricted aortic rings was assessed before and after washout after a 15-mm incubation with DPI at the indicated concentrations. Points are the means ± SE (n = 4-8). Right panel) DPI irreversibly inhibits ACh- but not nitroprussideinduced
relaxation
of rabbit
aorta.
Vasorelaxant
responses
elicited
by cumulative administrationof ACh, or sodium nitroprusside (SNP; inset), to norepinephrine-proconstricted aorticringswere assessed before (0) and after a 30-mm exposure to 1 iM DPI, either 15 mm (A) or 120 mm () after DPI washout. Points are means ± SE (n 4). of NO. from endothelial cells (33). DPI did prevent AChinduced vasorelaxation (Fig. 5). The effect was potent (IC,0 0.3 tiM; left panel) and irreversible (right panel). Although inhibition of vasorelaxation reached a plateau at - 55% after a single application of DPI (Fig. 5), exposure of aortic rings to a second application of 10 tM DPI 30 mm after the initial treatment resulted in 100% inhibition of relaxation.4 The basis of this incremental inhibitory effect is under study. In contrast to its effect on ACh-induced relaxation, DPI did not interfere with the ability of sodium nitroprusside to relax norepinephrmne-constricted aortic rings (Fig. 5, inset). Nitroprusside acts directly on vascular smooth muscle by generating NO. in situ (34, 35). Moreover, DPI did not attenuate the norepinephrine-induced vasoconstriction in the absence of ACh or nitroprusside.
I
+ +
DISCUSSION
FMN + RFLAV
+
ADPR
+
FUN
:
CH,
CH3
: io
NH, I
0
25
NO2-
+
I
I
50
75
NO3-, relative
I
100
%
Figure 4. Protection against irreversible inhibition by DPI. NADPH, NADP, NADH, or 2’,5’-ADPwere present at 5 mM during the 30-mm incubation of semipurified N0 synthase with DPI, whereas FAD, FMN, riboflavin (RFLAV), and adenosine 5-diphosphoribose (ADPR) were present at 0.5 mM. Each value is mean ± SD of triplicates.
IRREVERSIBLE INHIBITION
OF NO SYNTHASE
These findings bring to three the known families of inhibitors of NO. synthases. The inhibitors first described, N”substituted analogs of L-arginine (36, 37), have been extremely useful in demonstrating the participation of NO. synthesisin complex functions of intactcells,organs, and organisms. The most widely used analogs of this class, N’”methyl- and N”-nitro-L-arginine, display Kr’s in the low micromolar range and function as competitive, reversible inhibitors. The second class are calmodulin inhibitors, such as trifluoperazine, which block the calciumand calmodulindependent NO. synthase of cerebellum and endothelial cells (14),but have littleor no effecton macrophage NO. synthase.’ In comparison, DPI and its analogs are extremely potent inhibitors of NO. synthase activity in both macro-
O. A. Fasehun,
S. S. Gross,
and R. Levi, unpublished
observa-
tions.
‘N. S. Kwon, C. F. Nathan,
and D. J. Stuehr,
unpublished
obser-
vations.
101
phages and endothelial cells.The structuraldissimilarityof DPI and its irreversible effect suggest that its mechanism of action differs from that of either the L-arginine analogs or calmodulin inhibitors. In inhibiting macrophage NO. synthase activity, DPI appeared to affect a protein, as ‘complete inhibition remained after washing away of free DPI before assay. DPI inhibition was not due to itsreacting with added FAD or NADPH, as complete
inhibition
present
occurred
even
when
these
cofactors
were
excess over DPI, or alternatively, when the enzyme was treated with DPI and then washed before FAD and NADPH were added. Further, DPI did not alter the RPHPLC elution profiles of NADPH or FAD after coincubation under assay conditions. The macrophage protein that is inhibited by DPI may be immunologically inducible, because the activity of DPItreated NO synthase was not reconstituted when assayed in the presence of constitutive macrophage cytosolic proteins. The relevanttarget was not likelyto be a reductase that participates in BH4 regeneration, because BH4 was regenerated nonenzymatically with GSH in these experiments (25, 38). Of the cofactorsrequired by NO. synthase, only NADPH and FAD were able to block inhibition by DPI, and they did so only if present during DPI treatment. Of the FAD derivatives tested, FMN was the smallest fragment of FAD active in blocking the inhibition by DPI. The isoalloxazine ring and the ribitolphosphate were both essentialfor effective blocking. Both 2’,S-ADP and NADPH were effective antagonists, yet the closely related molecules adenosine 5-diphosphoribose and NADH were not. This structural specificity and lack of reaction of DPI with NADPH or FAD rule out the possibility that DPI inhibits NO. synthase by scavenging essential cofactors and instead suggest a mechanism by which DPI competes with both NADPH and FAD for specific binding sites on the protein target. DPI can undergo nucleophilic attack (30, 39); its covalent binding to discrete protein targets correlates with irreversible inhibition of neutrophil NADPH:O2 oxidoreductase (26) and mitochondrial NADH:ubiquinone reductase (29, 30). DPI was originally described as a hypoglycemic agent in rats (28). The hypoglycemia induced by DPI was later ascribed
to
in molar
its selective
inhibition
of mitochondrial
NADH:ubiqumnone
reductase in hepatocytes (28-30). More recently, DPI was shown to be a potent inhibitor of the NADPH:O2 oxidoreductase of neutrophils and macrophages (26, 27). Thus, both enzymes shown previously to be targets of DPI contain NADHor NADPH-utilizing flavoproteins. However, the susceptibility of such enzymes to inhibition by DPI varies widely. At one extreme, glutathione reductase was not inhibited after its coincubation with 50 ItM DPI for 30 mm at 37#{176}C.#{176} At the other extreme, the concentration of DPI required to irreversibly inactivate macrophage NO. synthase and NADPH:02 oxidoreductase (IC,o’s 0.05 and 1 tM [27], respectively)are 10- to 200-fold lower than those required to inhibit macrophage mitochondrial respiration (27). It may therefore be possible to use DPI to inhibit NO. synthesis and superoxide generation with relative selectivity. Endothelial cell NO. production was blocked by DPI without interfering with smooth muscle contraction. This suggests that smooth muscle ATP production was not markedly reduced, and illustrates the pharmacologic specificity of DPI. DPI, or structural analogs with greater specificity for NO. synthase, might prove useful in the study of hypotensive states induced
6D.
102
J.
Stuehr,
Vol. 5
unpublished
January
1991
observations.
by endotoxemia or the administration excessive production of NO. may (40).
of cytokines, in which play an important part
We thank Dr. Andrew Cross for providing DPI and its analogs and Dr. Owen Griffith for reviewing the manuscript. This work was supported by grants from the Leukemia Society of America (D. J. S.), the National Institutes of Health (CA 43610 [C. N.] and HL34215 [0. A. F., S. S. G., and R. L.]), and the Cancer Research Institute (C. N.). REFERENCES 1. Stuehr, D. biosynthesis:
J.,
and Marietta, M. A. (1985) Mammalian nitrate mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Nati. Acad. Sci. USA 82, 7738-7742 2. Hibbs, J. B., Jr., Taintor, R. R., and Vavrin, Z. (1987) Macrophage
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deiminase
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immno
nitrogen oxidation to nitrite. Science 235, 473-476 3. Palmer, R. M. J., Ferrige, A. G., and Moncada, 5. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.Nature (London) 327, 524-526 4. Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S. (1987) Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 61, 866-879
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The FASEB Journal
STUEHR ET AL.
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Production of an EDRF-like activity in the cytosol of NIE-l15 J., Kwon, N. S., and Nathan, C. F. (1990) FAD and neuroblastoma cells. FA SEB J. 4. 1494-1500 participate in macrophage synthesis of nitric oxide. Biochem. Biophys. Res. Commun. 168, 558-565 38. Kaufman, S. (1959) Studies on the mechanism of the enzymatic 26. Cross, A. R., and Jones, 0. T. G. (1986)The effectoftheinhibiconversion of phenylalanine to tyrosine. J. Biol. Chem. 234, tordiphenyleneiodonium on the superoxide generating system 2677-2682 of neutrophils. Specific labelling of a component polypeptide of 39. Banks, D. F. (1966) Organic polyvalent iodine compounds. the oxidase. Biochem. J. 237, 111-116 C/tern. Rev. 66, 243-266 27. Hancock, J. T., and Jones, 0. T. G. (1987)The inhibitionby 40. Kilbourn, R. G., Gross, S. S.,Jubran, A., Adams, J., Griffith, diphenyleneiodonium and itsanalogues of superoxidegenera0. W., Levi, R., and Lodato, R. (1990) N-methy1-L-arginine tion by macrophages. Biochem. J. 242, 103-107 inhibits tumor necrosis factor-induced hypotension: implica28. Holland, P. C., Clark, M. G., Bloxham, D. P., and Lardy, H. A. tions for the involvement of nitric oxide. Proc. Nati. Acad. &i. (1973) Mechanism of action of the hypoglycemic agent dipheUSA 87, 3629-3632 nyleneiodonium. J. BioL Chem. 248, 6050-6056 29. Gatley, S. J., and Sherratt, H. S. A. (1976)The effects of dipheReceived for publication August 9, 1990. nyleneiodonium on mitochondrial reactions. Relation of bindAccepted for publication September 26, 1990. GSH
IRREVERSIBLEINHIBITION
OF NO SYNTHASE
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