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(Received for publication, July 12, 1995; and in revised form, October 23,
1995) From the
The rapid and spontaneous interaction between superoxide
(O
It is becoming increasingly apparent that certain types of
inflammatory tissue injury are mediated by reactive metabolites of
oxygen and nitrogen. For example, it has been demonstrated that
administration of superoxide dismutase is effective at attenuating the
tissue injury observed in experimental models of arthritis, chronic gut
inflammation, and immune complex-induced pulmonary
injury(1, 2, 3) . Furthermore, models of
joint, bowel, and lung inflammation have been shown to be associated
with enhanced production of nitrogen oxides derived from the free
radical nitric oxide (NO)(4, 5, 6) . Indeed,
recent studies have demonstrated that inhibition of NO synthase also
provides substantial protection against the inflammatory tissue injury
observed in these models of acute and chronic
inflammation(4, 5, 6) . These data suggest
that both superoxide (O Recent
chemical studies have demonstrated that O This apparent ``antioxidant'' activity of NO has prompted
some investigators to suggest that the interaction between
O The reasons for
these apparent discrepant results are not clear. However, recent
evidence by Rubbo et al.(10) suggests that the
relative fluxes of the two free radicals may be an important
determinant as to whether NO enhances or inhibits
O
Nitric oxide was generated
using the spontaneous decomposition of the Sp/NO. Sp/NO spontaneously
decomposes at 37 °C and pH 7.4 at a known and constant rate (t
Hydroxylating activity of the various systems described
above was quantified by measuring the hydroxylation of BA to HB. At pH
7.4, the principal products of BA hydroxylation are the
monohydroxylated derivatives, 2-, 3-, and 4-hydroxybenzoate, with
2-hydroxybenzoic acid representing approximately 60% of total
product(32) . Fluorescence emission of the HB derivative
represents greater than 95% of total emission detected at 410 nm (i.e. for the three purified derivatives at equimolar
concentrations and physiological pH(32) ). 500-µl reaction
volumes containing 20 mM potassium phosphate buffer (pH 7.4)
and 0.15 M NaCl. 1.0 mM benzoic acid, 0.5 mM HX, and various concentrations of xanthine oxidase or Sp/NO were
incubated for 60 min at 37 °C. For some experiments, catalase (15
µg/ml) or superoxide dismutase (100 µg/ml) were included in the
reaction volumes, whereas in other experiments catalase and superoxide
dismutase were omitted and 5 µM Fe
Figure 1:
Oxidation of dihydrorhodamine to
rhodamine in the presence of constant superoxide production and various
fluxes of nitric oxide. All samples contained 15 µg/ml catalase and
were analyzed as described under ``Materials and Methods.''
Superoxide was generated by the HX/xanthine oxidase system at
Figure 2:
Interaction between NO and peroxynitrite.
NO was continuously produced by 50 µM Sp/NO in 50
mM potassium phosphate buffer (pH 7.4) at 37 °C and was
quantified using electrochemical detection. Peroxynitrite was dissolved
in ice-cold 0.1 N NaOH containing 0.1 mM diethylenetriaminepentaacetic acid. The arrows indicate
the time points at which a 1.00-µl addition of the base only or of
the alkaline peroxynitrite solution (containing 11 nmol) were
made.
Figure 3:
Oxidation of dihydrorhodamine to rhodamine
in the presence of constant nitric oxide production and various fluxes
of superoxide. All samples contained 15 µg/ml catalase and were
analyzed as described under ``Materials and Methods.'' NO was
produced at a flux of 1.0 nmol/min by Sp/NO in 20 mM potassium
phosphate buffer (pH 7.4) at 37 °C. Superoxide was generated by the
HX/xanthine oxidase system.
Figure 4:
Effect of superoxide on
peroxynitrite-mediated oxidation of dihydrorhodamine. Superoxide was
generated by dissolving 50 mg of KO
Fig. 5shows that neither O
Figure 5:
Effect of superoxide dismutase on the
oxidation of dihydrorhodamine to rhodamine in the presence of equimolar
fluxes of NO and O
Figure 6:
A, the effect of increasing NO flux on
O
The addition of catalase or
superoxide dismutase to solutions containing Fe
Figure 7:
Rhodamine production in the presence of
equimolar fluxes of NO and O
Figure 8:
A, hydroxylation of benzoic acid in the
presence of constant O
Fig. 9demonstrates that neither O
Figure 9:
Production of 2-hydroxybenzoate in the
presence of equimolar fluxes of NO and
O
Figure 10:
A, the effect of increasing NO flux on
O
Much of the vascular and tissue injury observed in certain
models of inflammation have been shown to be inhibited by either
superoxide dismutase or NO synthase inhibitors, suggesting that both
O Data obtained in the present study demonstrate
that in the absence of iron-catalyzed reactions, simultaneous
generation of equimolar fluxes of O We speculate that the decreased production of RH or HB in the
absence of iron and H Although the direct reaction of ONOOH with either NO or
O
Although reaction rates are not forthcoming from calculated
thermodynamic values, the possibility of the interaction of ONOOH with
NO or O Under conditions of limiting O When the same oxidation and hydroxylation experiments were performed
in the presence of O
The efficiency of such interactions could explain the results in Fig. 10A. Indeed it is well known that NO binds under
physiological conditions with ferrous heme containing compounds (e.g. hemoglobin and myoglobin), and moreover these reactions
are the chemical basis of current methodology used for NO
detection(39) . An alternative and more likely explanation for
this dramatic inhibitory effect of NO may be that NO shunts
O Our data confirm and extend
the results recently reported by Rubbo et al.(10) ,
who demonstrated that increasing fluxes of NO with respect to
O Two major physiological implications arise from our present study. (a) The role of ONOO Important consequences of the
oxidant/hydroxylation-dependent flux of NO and
O
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 40-47
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
) and nitric oxide (NO) to yield the
potent oxidants peroxynitrite (ONOO) and
peroxynitrous acid (ONOOH), has been suggested to represent an
important pathway by which tissue may be injured during inflammation.
Although several groups of investigators have demonstrated substantial
oxidizing and cytotoxic activities of chemically synthesized
ONOO
, there has been little information available
quantifying the interaction between O
and NO in the absence or the presence of redox-active iron. Using
the hypoxanthine (HX)/xanthine oxidase system to generate various
fluxes of O and HO and the spontaneous decomposition of the spermine/NO adduct to
produce various fluxes of NO, we found that in the absence of
redox-active iron, the simultaneous production of equimolar fluxes of
O and NO increased the oxidation of
dihydrorhodamine (DHR) from normally undetectable levels to
approximately 15 µM, suggesting the formation of a potent
oxidant. Superoxide dismutase, but not catalase, inhibited this
oxidative reaction, suggesting that O and not hydrogen peroxide (HO) interacts
with NO to generate a potent oxidizing agent. Excess production of
either radical virtually eliminated the oxidation of DHR. In the
presence of 5 µM Fe-EDTA to insure
optimum O
-driven Fenton chemistry, NO
enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As
expected, both superoxide dismutase and catalase inhibited this
Fe-catalyzed oxidation reaction. Excess NO production with respect to
O flux produced only modest inhibition
(33%) of DHR oxidation. In a separate series of studies, we found that
equimolar fluxes of O and NO in the
absence of iron only modestly enhanced hydroxylation of benzoic acid
from undetectable levels to 0.6 µM 2-hydroxybenzoate. In
the presence of 5 µM Fe-EDTA,
HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased
dramatically from undetectable levels to 4.5 µM of the
hydroxylated product. Superoxide dismutase and catalase were both
effective at inhibiting this classic
O
-driven Fenton reaction.
Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction
in a concentration-dependent manner such that fluxes of NO
approximating those of O and
HO virtually abolished the hydroxylation of
benzoic acid. We conclude that in the absence of iron, equimolar fluxes
of NO and O interact to yield potent
oxidants such as ONOO/ONOOH, which oxidize organic
compounds. Excess production of either radical remarkably inhibits
these oxidative reactions. In the presence of low molecular weight
redox-active iron complexes, NO may enhance or inhibit
O
-dependent oxidation and hydroxylation
reactions depending upon their relative fluxes.
) and NO are
important mediators of inflammation-induced tissue injury and
dysfunction. The mechanisms by which O and NO may either separately or in tandem mediate tissue injury
during inflammation remain the subject of active debate. and NO rapidly interact via a radical-radical reaction at a
diffusion-limited rate (k = 6.7 
10
M s
) to generate the
potent oxidant peroxynitrite (ONOO
)(7) .
Beckman and co-workers (8) have suggested that the interaction
between these two free radicals to yield ONOO
and its
conjugate acid, peroxynitrous acid (ONOOH), enhances dramatically the
toxicity of either O
or NO alone.
Indeed, it has been demonstrated in vitro using preformed or
chemically synthesized ONOO/ONOOH that these oxidants
are capable of directly oxidizing carbohydrates(8) ,
sulfhydryls(9) , lipids(10, 11) , and DNA
bases (12) as well as mediating bacteriocidal and endothelial
cell toxicity(13, 14) . It has also been demonstrated
that the simultaneous production of NO and O
by macrophages may result in the formation of
ONOO/ONOOH(15) . However, a series of recent
reports demonstrate that NO may actually inhibit
O
-dependent, iron (or
hemoprotein)-catalyzed lipid peroxidation in
vitro(16, 17, 18, 19, 20) . and NO is an important detoxification
pathway of potentially injurious
O-derived reactive oxygen metabolites
and thus may actually represent an endogenous anti-inflammatory
pathway. Indeed there is increasing evidence to suggest that NO may
protect cells and tissue against reactive oxygen metabolite-mediated
oxidative
damage(16, 17, 18, 19, 20) .
Wink et al.(17) have shown that exogenous NO protects
Chinese hamster lung fibroblasts, rat H4 hepatoma cells, and rat
mesencephalic dopaminergic cells against reactive oxygen
metabolite-induced cell injury. Assreuy et al.(16) recently demonstrated that although generation of NO
by activated macrophages is cytotoxic to Leischmania major,
simultaneous generation of NO and O or
addition of authentic ONOO failed to induce any
microbicidal activity. Finally, several investigators have demonstrated
that exogenous administration of NO inhibits ischemia-induced
microvascular dysfunction produced by
O
-dependent adherence and emigration of
neutrophils in the post capillary venules in
vivo(21, 22, 23) .-dependent, iron-catalyzed lipid
peroxidation (10) . In these studies the effects of NO were
assessed only in the presence of ferric iron. A recent preliminary
study from our laboratory suggests that the effects of NO on
O-dependent oxidative reactions may be
quite different depending upon whether redox-active transition metals
are present or absent(24) . Therefore, the objectives of the
present study were to: (a) systematically quantify the
oxidizing and hydroxylating activity of NO and/or
O in the absence or the presence of
redox-active iron and (b) characterize these reactions using
different fluxes of each radical. The physiological significance of our
findings is discussed.
Chemicals
Hypoxanthine (HX), (
)benzoic acid (BA), 2-hydroxybenzoic acid (HB), dimethyl
formamide, spermine, diethylenetriaminepentaacetic acid, and cytochrome c (horse heart) were purchased from Sigma. Potassium
superoxide (KO) was obtained from Pfaltz and Bauer, Inc.
(Waterbury, CT). Xanthine oxidase derived from bovine milk was supplied
by Calbiochem, and rhodamine 123 (RH) and dihydrorhodamine 123 (DHR)
were purchased from Molecular Probes, Inc. (Eugene, OR). The
spermine/NO adduct (Sp/NO) was a generous gift from Dr. Larry Keefer
(National Cancer Institute, Frederick, MD). Human recombinant
copper-zinc superoxide dismutase was obtained from Kabi-Pharmacia AB
(Uppsala, Sweden), and catalase was purchased from Boehringer Mannheim.
The tetramethylammonium salt of ONOO was synthesized
according to the method of Bohle et al.(25) . Briefly,
in a dry box
[Me
N][O]
(60 mg, 0.57 mmol) was transferred into a 100-ml side arm Schlenk flask
and capped up with clean septum. The Schlenk flask was then brought out
of the dry box, purged with dry nitrogen, and cooled to -78
°C in a dry ice/ethanol bath. Anhydrous ammonia (30 ml) is
then condensed into the system while keeping the flask at positive
pressure of nitrogen. A gas mixture of dry nitrogen and nitric oxide,
prepared by reduction of NaNO
(250 mg, 3.62 mmol) by
FeSO, was passed over PO
and NaOH
and very slowly bubbled through the tetramethylammonium superoxide
solution for 35 min, during which a deep orange-yellow color developed.
Excess nitric oxide and some ammonia are removed by gently warming the
flask under a stream of nitrogen.
[MeN][ONOO] was
isolated in quantitative yield as a bright yellow-orange lyophilized
powder by freezing the remaining solution with liquid nitrogen and
removing the ammonia in vacuo overnight. Sample purity was
ascertained by UV-visible spectra in water (pH 14), which showed only
the absorption for
[Me
][ONOO] at
302 nm (e = 1705 M cm
) and magnetic
susceptibility with a Faraday balance, which indicated that there were
no detectable paramagnetic (superoxide) impurities present in the
sample.
Generation of Superoxide, Hydrogen Peroxide, and/or
Nitric Oxide
Superoxide and hydrogen peroxide
(HO) were generated at 37 °C in a total
reaction volume of 500 µl containing 20 mM potassium
phosphate buffer (pH 7.4), 0.15 M NaCl, 0.5 mM HX,
and various concentrations of xanthine oxidase (0-10
milliunits/ml). The initial rates of O generation were determined spectrophotometrically by measuring
the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm and ranged from 0.25 to approximately 10 nmol/min at 37
°C(26) . We used relatively large concentrations of HX and
short incubation times in order to minimize the formation of urate and
thus its potential interference as a free radical scavenger as well as
to produce fluxes of O and
HO that were approximately equal(27) .
Hydrogen peroxide production by HX/xanthine oxidase at 37 °C was
determined using the peroxidase-catalyzed,
HO-dependent oxidation of p-hydroxyphenylacetic acid to yield its fluorescent product
2,2`-dihydroxybiphenyl-5,5`-diacetate(28) . We found that under
our conditions, 1 milliunit/ml xanthine oxidase catalyzed the formation
of approximately 1.0 nmol/min each of O and HO. = 39 min; (29) ) to yield 2
mol of NO/mol of adduct. Sp/NO solutions were prepared fresh each day
as a 10 mM stock solution in ice-cold 10 mM NaOH and
stored on ice until used. Various concentrations of Sp/NO (0-200
µM) were incubated at 37 °C in a total reaction volume
of 500 µl containing 20 mM potassium phosphate buffer (pH
7.4) and 0.15 M NaCl. The initial rate of NO generation via
Sp/NO decomposition at 37 °C for 50 and 100 µM Sp/NO
was determined electrochemically using a NO-specific electrode (World
Precision Instruments, Sarasota, FL) and was found to be 0.87 ±
0.13 and 2.36 ± 0.12 nmol NO/min, respectively. These values
agreed well with those calculated for 50 and 100 µM Sp/NO
(1.1 and 2.2 nmol NO/min, respectively) based upon the published t
= 39 min at 37 °C and pH 7.4 (29
Detection of Oxidizing and Hydroxylating
Species
Oxidant formation was detected using the
oxidant-sensitive nonfluorescent probe DHR, which when oxidized by two
electrons is converted to the highly fluorescent RH
product(30) . Preliminary data from our laboratory confirmed
previously published reports (31) demonstrating that only
potent oxidizing agents such as hydroxyl radical (OH),
ferrylhemoproteins, or ONOO
/ONOOH are capable of
oxidizing DHR to RH. Neither O
,
HO, nor NO (in the presence or absence of
O) oxidized substantial amounts of DHR in the time course
of our experiments. DHR was prepared as a 25 mM stock solution
in nitrogen-purged dimethyl formamide and kept stored in the dark at
-20 °C until used. A 5 mM stock solution of DHR was
prepared fresh each day by appropriate dilution of the 25 mM stock in dimethyl formamide. 500-µl reaction volumes
containing 20 mM potassium phosphate buffer (pH 7.4), 0.15 M NaCl, catalase (15 µg/ml), 0.05 mM DHR, 0.5
mM HX, and various concentrations of xanthine oxidase
(0-10 millunits/ml) or Sp/NO (0-200 µM) were
incubated for 30 min at 37 °C. The addition of catalase insured
that HO did not accumulate to levels that could
participate in the O-driven Fenton
reaction. For some experiments, catalase (15 µg/ml) or superoxide
dismutase (100 µg/ml) was included in the reaction volumes, whereas
in other experiments catalase and superoxide dismutase were omitted and
5 µM Fe-EDTA was included. The omission
of catalase and the addition of 5 µM
Fe
-EDTA allowed for optimum
O
-driven Fenton chemistry and
OH production. Following the 30-min incubation period,
reactions were terminated by dilution with 1.0 ml of cold
phosphate-buffered saline (pH 7.4). Rhodamine formation was quantified
using fluorescence spectroscopy in which an excitation wavelength of
500 nm and an emission wavelength of 536 nm were used. The
concentration of RH was then calculated using regression values (i.e. slope and intercept values) obtained from plots using RH
standards.
-EDTA
was included. Following the 60-min incubation period, reactions were
terminated by dilution with 0.5 ml of cold phosphate-buffered saline
(pH 7.4). Production of HB was quantified by measuring the fluorescence
obtained with excitation and emission wavelengths of 290 and 410 nm,
respectively. The concentration of HB was determined using HB
standards. All fluorescence emission measurements and spectra were
obtained using an Aminco/Bowman Series 2 luminescence spectrometer (SLM
Instruments, Inc., Rochester, NY).
Interaction between Peroxynitrite and Excess
Superoxide or Nitric Oxide
The ability of excess NO to
interact with chemically synthesized ONOO was
assessed using NO generated from Sp/NO and the tetramethylammonium salt
of ONOO
. Briefly, NO formation (1.0 nmol/min) was
continuously monitored using electrochemical detection (WPI NO
electrode) in the absence or the presence of a bolus addition of a
small aliquot of ONOO
(11 nmol) in a 2-ml reaction
volume containing 50 mM phosphate buffer. The rapid
disappearance of the NO signal signified the interaction between NO and
ONOO
. The interaction between excess
O
and ONOO was
determined by mixing a 10-fold molar excess of potassium superoxide
(KO
) prepared in ice-cold 0.1 N NaOH containing
0.1 mM diethylenetriaminepentaacetic acid with an alkaline
solution of the ONOO salt prepared as described
above. A small aliquot alkaline
O
/ONOO was
immediately (<1 min) added to a 1-ml reaction volume containing 50
mM potassium phosphate buffer (pH 7.4) and 50 µM DHR. Oxidation of DHR was compared with that when the same amount
of alkaline ONOO
was added. A decrease in the
oxidation of DHR was used as evidence to suggest that
O
decomposes ONOO.
Oxidation of Dihydrorhodamine in the Absence of
Iron
Oxidation of DHR to rhodamine 123 by different fluxes
of NO in the presence of a constant flux of O of 1.0 nmol/min is presented in Fig. 1. In the presence of
catalase and the absence of exogenous iron, the production of RH
increased sharply from normally undetectable amounts in the absence of
NO to a maximum concentration of 15 µM at fluxes of
NO approximately equal to that of O
(1.0 nmol/min). Increasing NO fluxes above 1.0 nmol/min produced
a decrease in DHR oxidation such that when the flux of NO approached
4-5 nmol/min, there was a 90% reduction in RH formation. The
addition of 200 µM spermine to reaction volumes producing
fluxes of superoxide and NO of approximately 1.0 nmol/min did not
inhibit rhodamine production. Subsequent studies revealed that addition
of ONOO to a solution of NO generated by the
decomposition of Sp/NO transiently reduced the NO signal, suggesting an
interaction between ONOO
and excess NO (Fig. 2). In a similar series of experiments performed in the
presence of catalase and the absence of exogenously added
Fe
and a constant flux of NO of 1.0 nmol/min, we
found that increasing fluxes of O
resulted in the production of RH in a pattern similar to that
produced above such that DHR oxidation was maximal when fluxes of NO
and O were approximately equal at 1.0
nmol/min for each (Fig. 3). Moreover, the yield of RH decreased
substantially with further increases in O flux such that DHR oxidation was inhibited by 75% when fluxes of
O reached a rate of 5.0 nmol/min
(5-fold that of NO formation) (Fig. 3). We found that excess
O may interact with ONOO and partially decompose this oxidant as measured by the ability
of O
to decrease the ability of
ONOO to oxidize DHR (Fig. 4).
1.0
nmol/min in 20 mM potassium phosphate buffer (pH 7.4) at 37
°C. Various fluxes of NO were achieved by increasing the
concentration of Sp/NO.
in ice-cold 0.1 N NaOH containing 0.1 mM diethylenetriaminepentaacetic
acid. Aliquots of this solution were then mixed with 35 nmol of
alkaline ONOO prepared as described in the legend to Fig. 2. Aliquots of ONOO
or ONOO
and O
were added to 50 µM of dihydrorhodamine in a 1.0-ml reaction volume containing 50
mM potassium phosphate buffer (pH 7.4) at 37 °C and
incubated for 30 min. RH production was quantified as described under
``Materials and Methods.''
nor NO generation alone at fluxes of 1.0 nmol/min in the
absence of exogenous Fe
-EDTA and the presence of
catalase was capable of producing more than 3.0 µM RH. In
contrast, approximately 12 µM RH was produced with the
simultaneous production of equimolar fluxes of
O
and NO. Under conditions where both
NO and O were present, the addition of
superoxide dismutase (0.1 mg/ml) inhibited RH production by more than
75%. The initial rate of RH production in the presence of 1.0 nmol/min
NO and 1.0 nmol O/min was almost
10-fold higher when compared with the rate achieved with a
O flux of 10 nmol/min (data not shown).
. Assays were
performed in the presence of 15 µg/ml of catalase as described
under ``Materials and Methods.'' The fluxes of both NO and
O were constant at 1.0 nmol/min.
Superoxide dismutase (SOD; 0.1 mg/ml) was added as described
under ``Materials and Methods.''
Oxidation of DHR in the Presence of
Fe
Oxidation of DHR by
different fluxes of NO in the presence of trace amounts of
Fe-EDTA
-EDTA and a continuous flux of
O
and HO (1.0
nmol/min for each) is shown in Fig. 6A. We found that
DHR oxidation increased from 15 µM in the absence of NO to
a maximum of 20 µM RH in the presence of
2.0
nmol NO/min. Interestingly, RH production was reduced by 40% when NO
production was increased further to
5 nmol NO/min. In a similar
set of experiments where O
and
HO were varied and NO production held constant
at 1.0 nmol/min, we found that oxidation of DHR increased sharply with
increasing O and HO fluxes and remained comparatively constant even when fluxes of
O and HO were
five times that of NO (Fig. 6B). We did observe a
modest inhibition (20%) of DHR oxidation when fluxes of
O and HO were
10 times that of NO (data not shown).
/HO-dependent
production of rhodamine in the presence of 5 µM
Fe-EDTA. Rhodamine was quantified as described under
``Materials and Methods.'' A constant flux of
1.0
nmol/min was used for both O
and
HO in all assays. B, the effect of
increasing O/HO flux on rhodamine production in the presence of 5 µM
Fe-EDTA. Rhodamine was quantified as described under
``Materials and Methods.'' NO flux was constant at
1.0
nmol/min in all assays.
-EDTA
and HX/xanthine oxidase attenuated RH production by >90% (Fig. 7). The generation of NO in Fe
-EDTA
solutions in the absence of O
and
HO resulted in production of very small amounts
RH (2.0 µM), whereas the simultaneous production of
O
/HO and NO in
the presence of Fe-EDTA oxidized approximately 13
µM DHR.
and in the
presence of 5 µM Fe-EDTA. Rhodamine was
quantified as described under ``Materials and Methods.'' NO,
O
, and HO were
all generated at 1.0 nmol/min each. Catalase (CAT; 15
µg/ml) or superoxide dismutase (SOD; 100 µg/ml) were
included in some reaction volumes.
Hydroxylation of Benzoic Acid in the Absence of
Iron
Hydroxylation of BA to yield HB with increasing fluxes
of NO in the presence of a continuous flux of
O (1.0 nmol/min) is shown in Fig. 8A. In the presence of catalase and the absence of
exogenous Fe-EDTA, we found that HB increased sharply
from normally undetectable amounts in the absence of NO to a maximum of
650 nM (650 pmol/ml) at fluxes of NO and
O
of 1.0 nmol/min each. Continued
increases in NO flux above 1.0 nmol NO/min attenuated hydroxylation
with complete inhibition of hydroxylation occurring at a flux of NO
that was four to five times that of O.
Using similar experimental conditions we found that increasing the flux
of O in the presence of continuous NO
generation (1.0 nmol/min) caused a comparable rise in benzoate
hydroxylation such that maximum hydroxylation occurred when fluxes of
each radical were 1.0 nmol/min (Fig. 8B). As the flux
of O increased with respect to NO
production, we observed a 50% inhibition of BA hydroxylation at fluxes
of O that were five times that of NO.
Complete inhibition was achieved when the flux of
O was 10 nmol/min or 10 times greater
than that of NO (data not shown). It should be noted that the absolute
amounts of HB (600-700 nM) produced by equimolar fluxes
of O and NO were in fact very small and
were close to the sensitivity limits of the assay.
production and
various fluxes of NO. Assays were performed as described under
``Materials and Methods.'' The data were obtained at a
constant O flux of 1.0 nmol/min.
All solutions contained 15 µg/ml catalase in 20 mM potassium phosphate buffer (pH 7.4) at 37 °C. B,
hydroxylation of benzoic acid in the presence of constant NO production
and various fluxes of O
. Assays were
performed as described under ``Materials and Methods.'' A
constant NO flux of 1.0 nmol/min was used. All solutions contained
15 µg/ml catalase in 20 mM potassium phosphate buffer (pH
7.4) at 37 °C.
nor NO alone were capable of mediating significant hydroxylation
in the absence of Fe-EDTA. Equimolar fluxes (
1.0
nmol/min) of each radical produced more than 510 nM HB, which
was inhibited by 60% by the addition of superoxide dismutase (0.1
mg/ml), suggesting that O
and NO
interact to yield an oxidant with only modest hydroxylating activity.
. The fluxes of NO and
O were constant at 1.0 nmol/min
each in all assays. Catalase (15 µg/ml) was also present in all
assays. Superoxide dismutase (SOD) was added as described
under ``Materials and Methods.''
Hydroxylation of Benzoic Acid in the Presence of
Fe
Fig. 10A illustrates BA hydroxylation with increasing amounts of NO in the
presence of Fe-EDTA
-EDTA and a continuous flux of
O
and HO of 1.0
nmol/min for each oxidant. Unlike the hydroxylation reaction in the
absence of iron and HO, production of HB
increased dramatically in the absence of NO such that hydroxylation
increased more than 6-fold in the absence of NO. We found that NO
inhibited the
O/HO-dependent
iron-catalyzed hydroxylation reaction in a concentration-dependent
manner such that as little as 4 nmol/min of NO virtually
eliminated the hydroxylation reaction (Fig. 10A).
Neither spermine nor NO
(data not shown)
significantly affected BA hydroxylation. When the NO flux was held
constant at 1.0 nmol/min and O
and
HO production were increased, we found a
concentration-dependent increase in hydroxylation of BA such that
maximum hydroxylation was obtained at fluxes of
O and HO of
approximately 2.0 nmol/min (Fig. 10B). Little
inhibition of HB formation was observed even when the
O/HO fluxes
were increased to 5.0 nmol/min (Fig. 10B).
/HO-dependent
hydroxylation of benzoate in the presence of iron. Assays were
performed as described under ``Materials and Methods.'' All
samples contained 5 µM Fe-EDTA, and
O
/HO fluxes
were held constant at 1.0 nmol/min in 20 mM potassium
phosphate buffer (pH 7.4) at 37 °C. B, the effect of
increasing O
and HO fluxes on O/HO and NO-dependent hydroxylation of benzoate in the presence of
iron. Assays were performed as described under ``Materials and
Methods.'' All samples contained 5 µM
Fe-EDTA, and the NO flux was constant at
1.0
nmol/min.
and NO are important mediators of
tissue injury and
dysfunction(1, 2, 3, 4, 5, 6) .
Because neither O nor NO are
particularly potent oxidants or cytotoxins, it has been suggested that
O and NO may combine to produce the
potent cytotoxic oxidants ONOO and ONOOH(8) .
Indeed, this hypothesis has generated tremendous interest because it
has provided a biochemical rationale to account for the remarkable but
perplexing protective effects of intravenous administration of L-arginine analogs (NO synthase inhibitors) or superoxide
dismutase in these pathophysiologic models of tissue injury and
inflammation(1, 2, 3, 4, 5, 6) .
Numerous studies have been published describing the physicochemical and
cytotoxic properties of chemically synthesized
ONOO
(8, 9, 10, 11, 12, 13, 14) .
However, there is a paucity of information quantitatively
characterizing the interaction between O
and NO under physiologic conditions. Thus, we have attempted to
systematically quantify the interaction between NO and
O in the absence or the presence of
redox-active iron. and
NO synergize to yield an oxidant or oxidants capable of oxidizing DHR
to RH ( Fig. 1and Fig. 3). Because catalase was present
throughout these experiments and because superoxide dismutase decreased
RH production by 90%, we propose that O but not HO nor OH interacts with NO to yield the oxidant or oxidants (Fig. 5). These data also confirm a previous report (31) that found that neither O
,
HO, nor NO per se is capable of
oxidizing substantial amounts of DHR in the absence of redox active
metals such as iron or hemoproteins. Only oxidants such as those
derived from Fenton-type reactions, ferryl hemoproteins, or
ONOO/ONOOH are potent enough oxidizing agents to
oxidize DHR. Indeed, decomposition of peroxynitrous acid to nitrate has
been suggested to proceed via a rate-limiting isomerization reaction
that yields a potent oxidizing agent capable of hydroxylating organic
substrates(8) . Thus, we also assessed the ability of
O
and NO to interact (in the absence of
iron and HO) to hydroxylate BA. We found a
similar pattern of hydroxylation of BA as observed for DHR oxidation in
that equimolar fluxes (1.0 nmol/min) of O and NO appeared to synergize to hydroxylate BA to HB (Fig. 8, A and B), although the magnitude of
this hydroxylation reaction was rather small (<15% that with iron
and HO present). Although we have not
definitely identified ONOO/ONOOH as the oxidants
produced in this system, we expect that this would be the likely
reaction pathway because of the rapid interaction between
O
and NO and because of the lack of
alternative explanations for the production of equally potent oxidants. O but in the presence of
either excess NO or O may be accounted
for on the basis of either secondary chemical interactions occurring
directly between NO or O and ONOOH. It
may also be due to the interaction between NO or
O with free radical intermediates of
DHR or BA to yield adducts with diminished fluorescence. The latter
possibility does not appear to be a major pathway because we did not
observe dramatic inhibition of DHR oxidation by excess NO in the
iron-containing system (Fig. 6A) nor did nitrosation of
HB by NO-derived nitrosating agents attenuate its fluorescence (data
not shown). The former hypothesis appears to be the more viable
explanation. has not been definitively
demonstrated, it has been suggested to be thermodynamically
possible(33, 34) . Koppenol et al.(34) have calculated Gibbs free energies (
G)
of -36 and -27 at neutral pH and 25 °C.
is at least indicated.
Therefore, competing reactions involving excess NO or
O with ONOOH could be a possible
mechanism for the decreased DHR oxidation and BA hydroxylation in the
absence of iron-catalyzed reactions. Albeit, our data indicate that
excess NO or O may be acting (at least
in our system) as modulators of the pro-oxidant characteristics of
ONOO/ONOOH, and by extension, we suggest that under
similar conditions in vivo, excess NO or
O
may act as an endogenous modulator of
ONOOH-mediated tissue damage. On the other hand, depending on the ratio
of fluxes of O to NO, oxidation and
hydroxylation reactions may be either enhanced or inhibited in the
absence of iron (Fig. 1, Fig. 3, and Fig. 10). flux,
excess NO will instead be auto-oxidized in the presence of molecular
oxygen-producing nitrogen oxides (e.g. NO,
NO
, or NO) that are not potent
oxidizing or hydroxylating agents but are potent N-nitrosating
agents (35) . We have recently demonstrated that
O will effectively inhibit NO-mediated N-nitrosation reactions(36) , and contrary to a recent
report(37) , we detected no significant change in xanthine
oxidase activity (measured via urate production) in the presence of 200
µM Sp/NO, which produces a flux of 4.0 nmol NO/min
(data not shown). Furthermore, the production of hydrogen peroxide from
xanthine oxidase was not inhibited by the presence of 200 µM SP/NO or 1 mM DEA/NO (data not shown). On the other hand,
increased production of O
at higher
xanthine oxidase concentrations is concomitant with increased urate
production (a potent free radical scavenger). Whereas urate-mediated
inhibition cannot be totally discounted, it apparently was not a
significant factor at O fluxes below
1.0 nmol/min, as indicated by the similarity in the shape of curves
shown in Fig. 1and Fig. 3. Moreover, total elimination
of possible urate interference was achieved with the use of pterin (500
µM) as substrate in place of HX, yet the data were
virtually identical to those shown in Fig. 3(data not shown). ,
HO, and 5 µM Fe-EDTA, qualitatively different results were
obtained. Generation of O
and
HO in the presence of Fe-EDTA
but not NO stimulated oxidation of DHR producing approximately 15
µM RH compared with the formation of 20 µM RH
in the presence of NO (1.0 nmol/min; Fig. 6A). These
data suggest that as the ratio of NO/O
increased from 0 to 2, oxidation of DHR increased by
approximately 30% (Fig. 6A). As the ratio was increased
further to 4.5, RH production was reduced by 40%. These data are
reminiscent of those reported by Rubbo et al.(10) using xanthine oxidase-dependent iron-catalyzed lipid
peroxidation. As expected, generation of O and HO (1.0 nmol/min each) in the
presence of iron increased production of HB tremendously (i.e. 4000 nM) (Fig. 10A). Remarkably, the
addition of NO to this iron-catalyzed hydroxylation system dramatically
inhibited hydroxylation of BA such that equimolar fluxes of NO
inhibited hydroxylation by 80% (Fig. 10A). Kanner et al.(38) recently suggested that NO may modulate
iron-mediated oxidative reactions by forming nitrosyl complexes with
ferrous iron or by the direct interaction of NO with
H
O. The sequence of reactions involving NO and
iron may proceed as follows:
away from iron-catalyzed
OH formation by the Fenton reaction and toward the
formation of an oxidant (e.g. ONOO
/ONOOH)
with only weak hydroxylating activity.
and HO modestly stimulated iron-catalyzed lipid peroxidation followed by
inhibition when fluxes of NO exceed those of
O. Furthermore, these same
investigators demonstrated that NO could partially inhibit
ONOO-induced lipid peroxidation(10) .
Interestingly, in our studies in the presence of iron, conditions under
which total inhibition of hydroxylation occurred resulted in only a
modest 33% inhibition of DHR oxidation ( Fig. 6versus 10), suggesting that NO is shunting O
away from iron-catalyzed OH formation and toward
the formation of a potent oxidant with weak hydroxylating activity.
at sites where both
O
and NO are produced (i.e. inflammatory foci) is dependent upon the relative fluxes of NO and
O in the extracellular space. Our data
suggest that excess production of one radical over the other may act as
an endogenous modulator of ONOO formation such that
the steady state levels of this potent cytotoxic oxidant never
accumulates above a certain amount. Indeed, the spontaneous
acid-catalyzed decomposition of another potent oxidant, hypothiocyanous
acid, is an example of autocatalytic regulation of oxidant
formation(40) . (b) NO may enhance or inhibit
oxidation and hydroxylation reactions depending upon the absence or the
presence of low molecular weight, redox-active metals such as iron.
Normally, there is little low molecular weight iron (e.g. amino acid, carbohydrate, or nucleotide chelates of iron) present
in most cells and tissue, with the vast majority of this metal
sequestered in its redox-inactive, protein-bound forms such as
ferritin- or transferrin-bound iron. However, it is known that certain
reductants (e.g. ascorbate and
O
) are very effective in releasing iron
from ferritin by reducing ferritin-bound Fe to
Fe
, which is no longer capable of being bound by the
protein(41, 42) . In addition to ferritin, there is
also a small but significant pool of low molecular weight iron chelate (e.g. non-protein-bound iron) located within cells. Studies by
Deighton and Hider (43) have identified this low molecular
weight iron chelate as a glutamate-iron complex (molecular weight of
1000-1500) that can easily exchange its iron with other more
potent chelators.
are temporal and spatial
considerations. To achieve the maximum oxidant resulting from NO/
O, the site orientation and timing of
the formation of these two species is crucial. The timing of the
superoxide production relative to the NO can be distinctly different in vivo and have a limited overlap under some immunological
and pathophysiological conditions. For instance, superoxide formation
of neutrophils reaches a flux 10 times higher than that of NO within
the first few minutes after treatment with phorbol ester(36) .
However, the flux of superoxide formed quickly subsides within an hour,
whereas the NO production continues for several hours. The time overlap
in which the flux of these two radicals is one to one is for a very
limited time; therefore the amount of peroxynitrite formed is small.
Conversely, cytokine-stimulated alveolar macrophage are thought to
generate both NO and O at the same
sustained rate for long period of time, implying that the oxidant
formed may be intentionally held high in this specific cell
line(15) . Yet, RAW macrophages appear to generate solely
nitrosating agents via the NO/O reaction without the
presence of superoxide (35) . This switching between oxidation,
hydroxylation, and nitrosation appears to be well orchestrated in the
immune response to pathogens and appears to be critical in host
defense. Although NO and superoxide can be generated from the same cell
type and cytokine influence, kinetic considerations must be carefully
examined to determine the reactive intermediates involved.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. A. Wink, J. A. Cook, S. Y. Kim, Y. Vodovotz, R. Pacelli, M. C. Krishna, A. Russo, J. B. Mitchell, D. Jourd'heuil, A. M. Miles, et al. Superoxide Modulates the Oxidation and Nitrosation of Thiols by Nitric Oxide-derived Reactive Intermediates. CHEMICAL ASPECTS INVOLVED IN THE BALANCE BETWEEN OXIDATIVE AND NITROSATIVE STRESS J. Biol. Chem., April 25, 1997; 272(17): 11147 - 11151. [Abstract] [Full Text] [PDF]
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C. Szabo, G. Ferrer-Sueta, B. Zingarelli, G. J. Southan, A. L. Salzman, and R. Radi Mercaptoethylguanidine and Guanidine Inhibitors of Nitric-oxide Synthase React with Peroxynitrite and Protect against Peroxynitrite-induced Oxidative Damage J. Biol. Chem., April 4, 1997; 272(14): 9030 - 9036. [Abstract] [Full Text] [PDF]
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HarryM. Lander, DavidP. Hajjar, BarbaraL. Hempstead, UroojA. Mirza, BrianT. Chait, S. Campbell, and LawrenceA. Quilliam A Molecular Redox Switch on p21ras. STRUCTURAL BASIS FOR THE NITRIC OXIDE-p21ras INTERACTION J. Biol. Chem., February 14, 1997; 272(7): 4323 - 4326. [Abstract] [Full Text] [PDF]
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S. Pfeiffer, A. C.F. Gorren, K. Schmidt, ErnstR. Werner, B. Hansert, D.S. Bohle, and B. Mayer Metabolic Fate of Peroxynitrite in Aqueous Solution. REACTION WITH NITRIC OXIDE AND pH-DEPENDENT DECOMPOSITION TO NITRITE AND OXYGEN IN A 2:1 STOICHIOMETRY J. Biol. Chem., February 7, 1997; 272(6): 3465 - 3470. [Abstract] [Full Text] [PDF]
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W. Aji, S. Ravalli, M. Szabolcs, X.-c. Jiang, R. R. Sciacca, R. E. Michler, and P. J. Cannon L-Arginine Prevents Xanthoma Development and Inhibits Atherosclerosis in LDL Receptor Knockout Mice Circulation, January 21, 1997; 95(2): 430 - 437. [Abstract] [Full Text]
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K. M. Miranda, M. G. Espey, K. Yamada, M. Krishna, N. Ludwick, S. Kim, D. Jourd'heuil, M. B. Grisham, M. Feelisch, J. M. Fukuto, et al. Unique Oxidative Mechanisms for the Reactive Nitrogen Oxide Species, Nitroxyl Anion J. Biol. Chem., January 12, 2001; 276(3): 1720 - 1727. [Abstract] [Full Text] [PDF]
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D. Jourd'heuil, F. L. Jourd'heuil, P. S. Kutchukian, R. A. Musah, D. A. Wink, and M. B. Grisham Reaction of Superoxide and Nitric Oxide with Peroxynitrite. IMPLICATIONS FOR PEROXYNITRITE-MEDIATED OXIDATION REACTIONS IN VIVO J. Biol. Chem., July 27, 2001; 276(31): 28799 - 28805. [Abstract] [Full Text] [PDF]
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