Production of Nitric Oxide by Mitochondria*
Cecilia
Giulivi
§,
Juan José
Poderoso¶, and
Alberto
Boveris
From the
Department of Molecular Pharmacology and
Toxicology, University of Southern California, Los Angeles,
California 90033 and the ¶ Laboratory of Oxygen Metabolism,
University Hospital and the
Laboratory of Free Radical
Biology, Department of Physical Chemistry, School of Pharmacy and
Biochemistry, University of Buenos Aires,
Buenos Aires 1113, Argentina
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ABSTRACT |
The production of NO· by mitochondria was
investigated by electron paramagnetic resonance using the spin-trapping
technique, and by the oxidation of oxymyoglobin. Percoll-purified rat
liver mitochondria exhibited a negligible contamination with other
subcellular fractions (1-4%) and high degree of functionality
(respiratory control ratio = 5-6). Toluene-permeabilized mitochondria,
mitochondrial homogenates, and a crude preparation of nitric oxide
synthase (NOS) incubated with the spin trap
N-methyl-D-glucamine-dithiocarbamate-FeII
produced a signal ascribed to the NO· spin adduct
(g = 2.04; aN = 12.5 G). The
intensity of the signal increased with time, protein concentration, and
L-Arg, and decreased with the addition of the NOS inhibitor
NG-monomethyl-L-arginine.
Intact mitochondria, mitochondrial homogenates, and submitochondrial
particles produced NO· (followed by the oxidation of
oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO· × (min·mg protein)
1, respectively, with a
Km for L-Arg of 5-7 µM.
Comparison of the rates of NO· production obtained with
homogenates and submitochondrial particles indicated that most of the
enzymatic activity was localized in the mitochondrial inner membrane.
This study demonstrates that mitochondria are a source of NO·,
the production of which may effect energy metabolism, O2
consumption, and O2 free radical formation.
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INTRODUCTION |
Nitric oxide
(NO·)1 is a free
radical generated in biological systems by nitric oxide synthases
(NOS). Because of its effect on neurotransmission, vasodilation, and
immune response (1-3), NO· plays an important role in
physiology, pathology, and pharmacology.
Studies with brain tissue and macrophage lysates have shown that NOS is
localized exclusively in the soluble fraction (3-6), and recent
studies have indicated that the majority (>80%) of bovine endothelial
NOS activity is bound to the particulate fraction of cell homogenates
(7, 8). Because the particulate fraction used in the studies was
expected to contain plasma membranes, as well as microsomes, and,
possibly, intracellular organelles, the actual subcellular location of
the activity remained to be determined. Other lines of evidence have
indicated the presence of NOS in the perinuclear region, in discrete
regions of the plasma membrane of cultured endothelial cells, and in
intact blood vessels (9, 10); immunocytochemical studies have revealed
the presence of a NOS, or an antigenically related protein, in
mitochondria isolated from different tissues (11-13). The predominant
association of this mtNOS with the mitochondrial membrane (11, 12), and its co-localization with succinate dehydrogenase, a mitochondrial marker of the inner membrane (13), suggested that this enzyme has a
particulate localization.
These studies as well as the presence of substrates and cofactors in
mitochondria required for NOS activity such as L-arginine (L-Arg), L-Arg transporters, Ca2+,
calmodulin, NADPH, and the availability of O2, led us to
postulate mitochondria as a potential source of NO·
production.
Following the use of a specific spin-trapping agent and the controlled
oxidation of oxymyoglobin, NO· production was detected in
purified mitochondrial preparations (intact mitochondria, permeabilized
mitochondria, mitochondrial homogenates, and submitochondrial
particles) and crude preparations of NOS (crude fraction) obtained from
rat liver.
Given the important implication of a mitochondrial production of
NO· for energy conservation mechanisms and free radical
production, such production may serve as the basis for a new
understanding of biochemical regulation, based on the ubiquitous
distribution of mitochondria and the diffusibility of NO·
through cellular membranes.
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MATERIALS AND METHODS |
Chemicals and Biochemicals
EDTA, EGTA, sodium succinate, sodium malate, sodium glutamate,
mannitol, ADP, sucrose, HEPES, bovine serum albumin (fatty-acid free),
and CHAPS were purchased from Sigma. Catalase, horseradish peroxidase
(grade I), and superoxide dismutase were obtained from Boehringer
Mannheim. The spin trap,
N-methyl-D-glucamine-dithiocarbamate-FeII
(MGD), was purchased from the Oklahoma Medical Research Foundation (Oklahoma City, OK). 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO or TEMPOL) was obtained from Aldrich. Oxymyoglobin was prepared
from 1 mM of commercially available horse heart myoglobin in 0.1 M Hepes buffer, pH 7.4 (degassed with helium), mixed
with a slight molar excess of sodium dithionite. The sample was
aerated, and excess dithionite was removed by gel filtration using a
Sephadex G-25 column (14).
Biological Materials
Liver mitochondria were isolated from adult Wistar rats
(180-200 g) by differential centrifugation, essentially as described in Ref. 15. The livers from rats (anesthetized with pentobarbital or
decapitated) were excised, washed with 0.25 M sucrose, and homogenized (1/10, w/v) using MSHE (0.22 M mannitol, 0.07 M sucrose, 0.5 mM EGTA, 0.1% bovine serum
albumin (fatty-acid free), 2 mM Hepes/KOH, pH 7.4) at
4 °C. The homogenate was centrifuged at 600 × g for
5 min in a JA-17 rotor. The supernatant was centrifuged at 10,300 × g for 10 min. Intact, purified mitochondria were isolated using Percoll to remove contaminating organelles and broken
mitochondria (16). Percoll was selected as the gradient medium because
of its negligible osmolarity and chemical inertness (17, 18). The
pellet (mitochondrial fraction) was resuspended in 5 ml of MSHE
supplemented with 20 ml of 30% Percoll in 225 mM mannitol, 1 mM EGTA, 25 mM Hepes, 0.1% bovine serum
albumin (fatty-acid free). This solution was spun at 95,000 × g in a Beckman Ti60 rotor for 30 min. The fraction with a
density of 1.052-1.075 g/ml was collected and washed twice with MSHE
at 6,300 × g for 10 min to remove the Percoll.
Purified mitochondria were washed twice with 150 mM KCl,
followed by two washings with MSHE. Permeabilized mitochondria were
prepared using a controlled treatment of purified mitochondria with
toluene (19). Mitochondria were resuspended to 20 mg of protein/ml in
MSHE supplemented with 8.5% (w/v) polyethylene glycol 8000 at
0-4 °C. Toluene (0.2%, v/v) was added to the suspension, which was
gently inverted every 15-30 s over a 2-min period. Mitochondria were
then pelleted by centrifugation at 15,000 × g for 4 min at 4 °C, and the supernatant was removed by aspiration.
Mitochondria were washed twice, and resuspended, in MSHE containing
polyethylene glycol. Submitochondrial particles (SMP) were obtained by
sonication (20) of purified mitochondria and stored at
20 °C at a
protein concentration of 20 mg/ml. Mitochondrial homogenates were
obtained by mechanical homogenization of the mitochondrial fraction
subjected to three cycles of freeze-thawing. A crude fraction of NOS
was obtained from purified rat liver mitochondria. Mitochondria from two to four livers were homogenized with Buffer A (1 mM
EDTA, 5 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 50 mM Hepes, pH 7.5). This
homogenate was centrifuged at 150,000 × g for 1 h
at 0-4 °C. The mitochondrial membranes were washed with Buffer B
(Buffer A plus 1 M KCl, 10% glycerol) and centrifuged at
the same speed for 30 min. The pellet was treated with Buffer A plus 20 mM CHAPS at 4 °C under continuous stirring. After 30 min, the suspension was centrifuged at 150,000 × g for
30 min. This supernatant, concentrated in a CentriconTM cartridge
(Mr cut-off of 30,000; Amicon, Danvers, MA), was
referred to as crude fraction (150,000 × g
supernatant).
Detection of Nitric Oxide
Electron Paramagnetic Resonance Assay--
Aliquots of the
samples, containing the NO· spin trap MGD (21, 22), were
transferred to bottom-sealed Pasteur pipettes, and the spectra were
recorded at 22 °C with an X-band EPR spectrometer, operating at 9.77 GHz. Instrument settings are described in the legend for Fig. 1. The
settings were selected using either sodium nitroferricyanide or
nitrosoglutathione as NO·-releasing agents.
Oxymyoglobin Spectrophotometric Assay--
The oxidation of
oxymyoglobin (23) was followed at 581-592 nm (
581-592 = 11.6 mM
1 cm
1) with a
double-beam spectrophotometer Hitachi U-3110 with a multiple wavelength
program at 22 °C using 50 µM oxymyoglobin. Superoxide dismutase (1 µM) and catalase (1 µM) were
added to prevent interference by O
2 and
H2O2, respectively, that might be produced by
the mitochondrial respiratory chain or by autoxidation of
tetrahydrobiopterin, heme, or flavin semiquinones. The reaction
medium used with SMP, mitochondrial homogenates, or crude fraction
consisted of 1 mM L-Arg, 1 mM
magnesium acetate, 1 mM CaCl2, 0.1 mM NADPH, and 12 µM tetrahydrobiopterin in
0.1 M Hepes buffer, pH 7.5. The reaction medium used with
intact mitochondria consisted of 10 mM succinate, 225 mM sucrose, 5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 50 µM oxymyoglobin, and 20 mM Hepes/KOH, pH 7.4. The reaction medium used with permeabilized mitochondria consisted of
225 mM sucrose, 5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 0.1 mM NADPH, 1 mM CaCl2, 10 µM reduced tetrahydrobiopterin, 20 mM
Hepes/KOH, pH 7.4, plus 8.5% (w/v) polyethylene glycol 8000.
Protein Determination
Protein was determined by the Lowry assay (24) using bovine
serum albumin as standard.
Data Evaluation
All assays were done in duplicate and were repeated five to
eight times in separate experiments using 2-4 rats/experiment. Data
are presented as mean ± S.E., in which the S.E. were between 10 and 12% of the mean values.
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RESULTS |
Assessment of Purity and Functionality of Rat Liver
Mitochondria--
Rat liver mitochondria were isolated by differential
centrifugation (15), purified by Percoll centrifugation (16), and washed with high ionic strength solutions. This procedure allowed the
efficient removal of contaminating organelles, broken mitochondria, arginase (25), and adsorption artifacts, yielding a highly purified preparation. This is supported by the low degree of non-mitochondrial contamination (1-4%; Table I), which
was comparable with, and in some cases less than, that obtained with
other purification procedures (18, 26, 27). Mitochondria isolated using
this procedure differ from those obtained by differential
centrifugation in that the former exhibited a higher respiratory
control ratio, indicating functional integrity and membrane
intactness (Table II).
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Table I
Enzymatic characterization of isolated rat liver mitochondria
Cytochrome c oxidase activity was expressed as nanomoles of
cytochrome c oxidized/min/mg of protein;
glucose-6-phosphatase, 5'-nucleotidase, and acid phosphatase activities
were expressed as nanomoles of inorganic phosphate/min/mg of protein;
catalase activity was expressed as micromoles of H2O2
consumed/min/p mg of protein (see Ref. 26 and references therein).
Recovery is the ratio of specific activities of an enzyme in
mitochondria and in the homogenate (Ratio) corrected by the protein
recovered in the mitochondrial fraction (3%).
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Table II
Comparison of mitochondrial functional parameters
Mitochondria from rat liver (n = 6) were prepared by
differential centrifugation (differential centrifugation; Ref. 15) and
purified by Percoll (16) followed by two washings with high ionic
strength solution (Percoll centrifugation; Ref. 25). Respiration in the
absence (State 4) or in the presence (State 3) of 0.2 mM
ADP was measured polarographically by using a Clark-type oxygen
electrode in a medium containing 0.22 M sucrose, 50 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, in 10 mM Hepes buffer, pH 7.4, with 10 mM sodium
succinate or 5 mM glutamate, 0.5 mM malate. The
temperature was 25 °C. RCR is defined as the State 3/State 4 respiration ratio (28) and P/O as ATP formed/O2 consumed (29);
ND, not determined.
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Detection of Nitric Oxide in Mitochondrial Preparations by
Spectroscopic Techniques: Detection of NO· by Spin
Trapping/EPR--
Toluene-treated mitochondria with an increased
permeability for the spin trap and external NADPH were incubated with
the spin trap ((MGD)2/FeII) for 1 h at
room temperature. A weak EPR signal consisting of a triplet
(aN = 12.5 G; giso = 2.04; intensity ratio 1:1:1; Fig.
1A) was assigned to the
(MGD)2/FeII-NO· complex (21, 22) by
comparison with the signal obtained with the NO· donor,
nitrosoglutathione (Fig. 1E). In addition to the triplet, a
line from a quartet signal was present assigned to the
(MGD)2/CuII complex produced by the reaction of
free CuII, in the homogenate or in the reaction solution,
with the excess of MGD. The addition of L-Arg increased the
signal by 30% (Fig. 1B), whereas
NG-monomethylarginine (NMMA), the
competitive inhibitor of NOS (30), decreased the signal by 50% (Fig.
1C) and 20% (Fig. 1D) in the absence and
presence of L-Arg, respectively. Based on its sensitivity to NMMA, the formation of the
(MGD)2/FeII-NO· signal in the biological
sample without L-Arg suggests an endogenous pool of Arg
capable of sustaining NO· production through an NOS-catalyzed
reaction (Fig. 1C). The NMMA-insensitive EPR signal
suggested the presence of a labile pool of
NO·.2 Of note, the
addition of 5 µg/ml calmodulin and/or 1 mM
Ca2+ did not significantly affect the signal intensity,
indicating that NOS was fully active with the cofactors present in our
preparations.

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Fig. 1.
EPR spectra of permeabilized mitochondria
supplemented with NOS substrates and inhibitors after 1 h of
incubation. The reaction medium contained 225 mM
sucrose, 5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 0.1 mM NADPH, 1 mM CaCl2, 10 µM reduced
tetrahydrobiopterin, 20 mM Hepes/KOH, pH 7.4 plus 8.5% (w/v) polyethylene glycol 8000, 30 mM
(MGD)2/FeII, and (5.2 mg of protein)
toluene-treated mitochondria. These samples were incubated for 1 h
at room temperature without (A and C) and with
(B and D) 1 mM L-Arg,
plus 10 mM NMMA (C and D). The EPR
spectra of the iron-nitrosyl complex ( ) or the
(MGD)2-CuII complex ( ) were recorded at room
temperature operating at 9.77 GHz in an EPR spectrometer (Bruker
ECS106). Instrument settings: modulation frequency, 100 kHz; modulation
amplitude, 2.9 G; sweep scan 0.9 G/s; sweep width, 150 G; microwave
power, 100 milliwatts; time constant, 0.6 s; receiver gain,
2.5 × 105. EPR spectrum of the
(MGD)2/FeII-NO· complex (E) originated
from a reaction mixture containing 0.1 mM
nitrosoglutathione, 30 mM
(MGD)2-FeII, 0.5 M Hepes buffer, pH
7.4. Nitrosoglutathione was synthesized in situ by the
addition of 0.2 mM sodium nitrite in 0.1 M HCl
and 0.2 mM reduced glutathione in 1 M HEPES
buffer, pH 7.4. Spectrum E was recorded with a receiver gain
of 3.2 × 103, and a time constant of 0.2 s. All
the EPR spectra were recorded in bottom-sealed Pasteur pipettes.
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Toluene-treated rat liver mitochondria, incubated with the spin trap
for 8 h to increase the substrate and inhibitor concentrations in
the mitochondrial matrix, exhibited signal intensities 2.3- and
2.7-fold higher than those found at 1 h, with and without L-Arg addition, respectively (Fig.
2, A and B).
Preincubation of mitochondria with NMMA inhibited the signal formation
by 50% and 15% in the absence and presence of L-Arg,
respectively (Fig. 2, C and D). A weak
(MGD)2/FeII-NO· signal was also noted in
the absence of mitochondrial homogenate (data not shown); this signal
is likely to originate from NO· diffusion from ambient air
(about 0.1 ppm).

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Fig. 2.
EPR spectra of permeabilized mitochondria
supplemented with NOS substrates and inhibitors after 8 h of
incubation. Toluene-permeabilized mitochondria (5.2 mg of protein)
were added to reaction mixtures without (A and C)
and with (B and D) 1 mM
L-Arg, plus 10 mM NMMA (C and
D) and then incubated for 8 h at room temperature. The
EPR conditions and reaction mixtures were described under Fig. 1.
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Mitochondrial homogenates or a crude fraction of NOS incubated for
1 h with L-Arg and the spin trap showed the same
three-line EPR spectrum observed with toluene-permeabilized
mitochondria (Table III). The EPR signal
intensities obtained with crude fraction were 5-7-fold higher than
those observed with permeabilized mitochondria. The signal intensities
were increased by L-Arg supplementation and decreased by
NMMA addition to different extents, depending on the specific
biological preparation (Table III). Similar results were obtained using
L-N5-(1-iminoethyl)ornithine
(NIO), another NOS inhibitor (data not shown). The high concentrations
of NMMA required in these experiments are indicative of the competitive
kinetics of the inhibition of NOS by NMMA in the presence of an
endogenous pool of L-Arg, the latter most likely sustained
by proteolytic activities present in the samples.
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Table III
EPR signal intensities of the (MGD)2FeIINO· for
different mitochondrial preparations
Toluene-permeabilized mitochondria, mitochondrial homogenates, or crude
fraction were incubated for the indicated times using 1 mM
L-Arg and 1 mM or 5 mM (number
between parentheses) of NMMA. The EPR signals were recorded using the
instrumental settings described under Fig. 1 legend, and the signal
intensities were expressed relative to no additions in toluene-treated
mitochondria. The height intensities were evaluated from the signal
peak at low magnetic field.
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The iron-nitrosyl complex signal intensities obtained with
toluene-treated rat liver mitochondria after 1 h of incubation showed a linear dependence on protein concentration. Double integration of the EPR signals and interpolation of calibration curves of the area
of the low-field peak versus TEMPO or TEMPOL allowed the
quantification of the EPR signals. This quantification permitted the
calculation of an NO· production rate of 48 ± 2 nmol/mg
mitochondrial protein (r = 0.99). The NO·
experimentally detected by EPR accounted for approximately 16% of the
rate of metmyoglobin formation, even when an excess of spin trap was
used. This underestimation may be due to the metabolism of the spin
trap by mitochondrial preparations to EPR silent species, probably of
the type (MGD)2FeII-NO·-X,
where X = halogen ions or NO2, as has been
described for diethyldithiocarbamate (31, 32). This notion is
strengthened by the similar recovery (12%) of an EPR signal of a
synthetic iron-nitrosyl complex (formed by incubating sodium
nitroprusside, an NO· donor, and the spin trap) and the decrease
in the signal intensities with protein concentrations above 12 mg.
Measurement of NO· Production by the Oxidation of
Oxymyoglobin--
The production of NO· in the presence of
L-Arg was measured in intact mitochondria, mitochondrial
homogenates, and submitochondrial particles by following the
NMMA-sensitive oxidation of oxymyoglobin to metmyoglobin (Fig.
3). The NMMA-insensitive rates of
oxymyoglobin oxidation were 10-20% of the total rate of metmyoglobin
formation using mitochondria and mitochondrial homogenates, and 30%
when using SMP. The higher unspecific oxidation in the latter instances may be attributed to a direct oxidation of oxymyoglobin by a component of the respiratory
chain.3

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Fig. 3.
NO· production by mitochondrial
homogenates, submitochondrial particles, and intact mitochondria.
NO· production by intact, purified mitochondria ( ) was
measured in a reaction mixture containing 0.225 M sucrose,
5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 10 mM succinate, 1 mM L-Arg, 20 mM Hepes/KOH, pH 7.4 supplemented with 50 µM oxymyoglobin. Mitochondrial
homogenates ( ) or SMP ( ) were incubated in 1 mM
L-Arg, 1 mM
MgCl2H3O2, 1 mM
CaCl2, 0.1 mM NADPH, 12 µM
tetrahydrobiopterin, in 0.1 M Hepes buffer, pH 7.5., supplemented with 50 µM oxymyoglobin. The transition oxy
[ ] metmyoglobin was followed at 581-592 nm during the first 3 min. Parallel experiments were performed in samples preincubated with
10 mM NMMA.
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The rates of NO· production increased linearly with the protein
concentration of mitochondrial preparations (Fig. 3). The specific rates were 1.36, 4.9, and 7.1 nmol × (min·mg
protein)
1 for intact mitochondria, mitochondrial
homogenates, and submitochondrial particles, respectively. The
activities obtained with mitochondrial homogenates and submitochondrial
particles were higher than those obtained with intact mitochondria
because the former were assessed under conditions for optimal NOS
activity. A comparison of the rates of SMP and mitochondrial
homogenates under identical conditions indicated that most of the
activity was detected in the mitochondrial inner membrane (considering
that 30% of the rat liver mitochondria protein corresponds to the
inner membrane fraction (33), 25-35% of the particles had the
"right side-in" conformation (34), and 80% of the activity
(experimentally determined) was recovered after the sonication
procedure) suggesting that NOS could be mainly (60-80%) localized in
this membrane fraction.
The production of NO· by intact mitochondria was followed by the
NMMA-sensitive oxidation of oxymyoglobin in the presence of L-Arg (Fig. 4A).
The rapid onset of the production was indicative of a fast transport of
L-Arg into mitochondria, consistent with the reported
L-Arg carriers found in isolated mitochondria from different tissues (35-37). The rate of NO· production by
mitochondria yielded a classic hyperbolic response saturated with
L-Arg concentrations above 20 µM (Fig.
4A). The apparent Km for
L-Arg was 5 µM (1.1 nmol/mg of protein) and
the Vmax was 0.38 µM/min
calculated from double-reciprocal plots (Fig. 4A,
inset). These kinetic constants were consistent with those
observed for permeabilized mitochondria (Vmax = 5 nmol of NO·/min/mg of protein, and Km for
L-Arg of 7 µM; Fig. 4B), as well
as with those reported for brain homogenates (as sources of nNOS)
during the conversion of L-Arg to citrulline
(Km = 6 µM for Arg and
Vmax = 0.15 µmol/min/mg protein; Ref. 38). The
Km for L-Arg was 20-50 times lower than
the matrix concentration of L-Arg (150-300
µM; Refs. 39 and 40), a value within the normal range of
L-Arg in rat liver (20-50 nmol/g wet weight; Ref. 25),
when expressed per gram of liver wet weight (38 nmol/g wet weight).
These concentrations indicate that mtNOS could be functionally active
based on the requirements of L-Arg availability under
normal conditions, and that if the kinetic properties of mtNOS in
intact mitochondria are similar to those in vivo, it is
unlikely that Arg levels play an important role in the regulation of
mtNOS.

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Fig. 4.
NO· production by intact, coupled
mitochondria and toluene-permeabilized mitochondria. Intact,
coupled mitochondria (panel A) or toluene-permeabilized
mitochondria (panel B) at 1 mg of protein concentration were
incubated in the respective reaction mixtures (0.225 M
sucrose, 5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 10 mM succinate, 20 mM Hepes/KOH, pH 7.4, for mitochondria, and 225 mM sucrose, 5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, 0.1 mM NADPH, 1 mM CaCl2, 10 µM reduced tetrahydrobiopterin, 20 mM
Hepes/KOH, pH 7.4, plus 8.5% (w/v) polyethylene glycol 8000 for
permeabilized mitochondria). All incubations contained 50 µM oxymyoglobin (see "Materials and Methods").
Inset, double-reciprocal plot of the rates of NO·
production and L-Arg concentration. Data were obtained from
the respective main figures.
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A slow rate of NO· production by intact mitochondria was also
detected in the absence of added L-Arg (11 nM/min; Fig. 4A). This rate may reflect a slow
but constant production of NO· by mitochondria using endogenous
L-Arg, which may be increased under special
pathophysiological conditions, such as those entailing an elevation of
Ca2+.
The NO· production by intact mitochondria was not altered by
D-Arg addition, was partially inhibited by NIO, and was
totally inhibited by NMMA (Table IV). The
lack of effect by the addition of D-Arg may be explained by
the selectivity of the Arg transporter for the L-isomer,
and the partial inhibition by NIO can be accounted for by a limited
permeability of the inhibitor. Consistent with this possibility is a
noted increase in the inhibition of of NO· production by the
pre-incubation of mitochondria with NIO (Table IV).
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Table IV
Inhibition of NO· production by intact mitochondria
Intact rat liver mitochondria (0.52 mg of protein/ml) were incubated in
1 ml of the reaction mixture containing 30 µM
L-arginine and 50 µM oxymyoglobin. The
suspensions were supplemented with 0.1 mM inhibitors, and
the rate of NO· production was quantified by measuring the oxidation
of oxymyoglobin at 581-592 nm after 5 min of incubation. Mitochondria
were preincubated with the inhibitors for 10 min, and then
L-Arg was added.
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In similar experiments, preincubation (10-15 min) of SMP with 5 mM NMMA was found to inhibit the endogenous production of NO·, whereas the addition of L-Arg reversed such
inhibition (Table V).
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Table V
Production of NO· by submitochondrial particles
SMP (0.4 mg of protein) were incubated in 0.1 M HEPES
buffer, pH 7.4, for 15 min with 5 mM NMMA, and the reaction
started by L-Arg addition. The rate of NO· production was
measured in the presence of 50 µM oxymyoglobin, and the
formation of metmyoglobin was followed spectrophotometrically at
581-592 nm. Specific activities were calculated as total activity
minus the activity in the presence of NMMA. Other conditions are
explained under "Materials and Methods."
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DISCUSSION |
The following lines of evidence offer support for the
mitochondrial generation of NO·.
First, the negligible contamination of the mitochondrial preparations
with other subcellular fractions (Table I) and the integrity and
functionality of these preparations (Table II) support the production
of NO· by mitochondria.
Second, the production of NO· by mitochondria was demonstrated
by two different spectroscopic assays: the formation of metmyoglobin, and spin trapping/EPR. To rule out species other than NO·
reacting with oxymyoglobin (41-45), the rate of oxidation of
oxymyoglobin was monitored under controlled conditions (with catalase
and superoxide dismutase and measuring sensitivity to NMMA). However,
because some NO·-derived oxides may still be able to produce
this reaction, unequivocal identification of NO· was furnished
by using the spin trap MGD, the iron-nitrosyl complex ESR signal of
which is considered a "fingerprint" of NO· (21, 22). The
advantages of this technique (selectivity of the spin trap toward
NO· and the lack of toxicity of the spin trap in biological
systems) were initially limited by the free access of the spin trap to the biological source, the low recovery of the iron-nitrosyl adduct, and the presence of quenchers of NO· (e.g.
hemoproteins, [Fe-S] clusters) that effectively compete with the spin
trap MGD. These limitations were overcome by using millimolar
concentrations of the spin trap (to effectively compete with other
possible NO· quenchers) with toluene-treated mitochondria and
mitochondrial homogenates (to allow free access of the spin trap to the
biological source of NO·). The use of submitochondrial particles
and mitochondrial homogenates, albeit less physiologically relevant
than intact mitochondria, devoid of the limitations noted above
permitted the selection of optimal conditions for NO· production
by mitochondria.
Third, the production of NO· is catalyzed by an enzyme, likely a
NOS isoform, located at the mitochondrial inner membrane. This was
inferred from three separate lines of evidence. (i) NO·
production was modulated by NOS substrates (L-Arg) and
inhibitors (NMMA, NIO, and D-Arg); (ii) the rate of
NO· production by mitochondria and SMP versus
L-Arg concentration followed a similar pattern to that
described for NOS purified from different tissues; (iii) the higher
specific activities in SMP or crude fraction (about 2 and 10 times
higher, respectively) than those obtained with mitochondrial
homogenates or permeabilized-mitochondria were indicative of an enzymic
activity located at the inner membrane. Conclusive evidence that a NOS
isoform was responsible for the NO· production was provided by
the purification and characterization of the enzyme from purified rat
liver mitochondria reported in the accompanying paper (46).
The rate of NO· production by rat liver mitochondria reported
herein and by others4 is
similar to that of O
2 (about 1.2 nmol of O
2/min/mg of
protein, equivalent to 0.6 nmol of H2O2/min/mg
of protein; Ref. 33). At saturating concentrations of
L-Arg, a steady-state concentration of NO· in the
range of 0.1-0.5 µM may be sustained. These values
contemplate the reaction of NO· with cytochrome oxidase or with
O2 as the main catabolic pathways. Of note, this level of
NO· is biologically relevant because it is in the range of those concentrations reported to inhibit respiration in synaptosomes (47) and
intact mitochondria (48). Given the role of NO· as cellular
messenger, transmitter, and regulator (1-3), it could be hypothesized
that this inhibition (or modulation) of mitochondrial respiration by
NO· may represent a novel biochemical pathway regulating the
supply of O2 and energy to tissues under dynamic
conditions.
 |
ACKNOWLEDGEMENT |
We thank Dr. Enrique Cadenas for thoughtful
comments on this manuscript.
 |
FOOTNOTES |
*
This work was supported by the University of Southern
California Liver Disease Research Center (Grant P30DK485222 from the National Institutes for Health) and by National Science Foundation Grant MCB 9724060. These results were presented in part at the Annual
Meeting of The Oxygen Society, November 21-25, 1996, Santa Barbara,
CA.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 and reprint requests should be addressed:
Dept. of Molecular Pharmacology and Toxicology, University of Southern
California, 1985 Zonal Ave., Los Angeles, CA 90033. Tel.: 213-342-1420;
Fax: 213-224-7473; E-mail: cgiulivi{at}hsc.usc.edu.
1
The abbreviations used are: NO·, nitric
oxide; MGD-Fe,
N-methyl-D-glucamine-dithiocarbamate-FeII
complex; NMMA,
NG-monomethyl-L-arginine;
NIO,
L-N5-(1-iminoethyl)ornithine;
TEMPO or TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; mtNOS,
mitochondrial nitric oxide synthase; NOS, nitric oxide synthase; SMP,
submitochondrial particles; EPR, electron paramagnetic resonance; G,
gauss.
2
C. Giulivi, unpublished observations.
3
C. Giulivi and E. Cadenas, unpublished
observations.
4
C. Richter, personal communication.
 |
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