| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Institut National de la Santé et de la Recherche Médicale (INSERM) U408 and IFR 02, Faculté X. Bichat, Paris, France;
Laboratory of Oxygen Metabolism, University Hospital, University of Buenos Aires, Argentina; and
§ Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
1Correspondence: INSERM U408, Faculté X. Bichat, BP416, 75870 Paris Cedex 18, France. E-mail: jbb2{at}bichat.inserm.fr
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: sepsis • respiratory insufficiency • respiratory muscles • mitochondria • nitric oxide • oxidative stress • nitrotyrosine
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. An impaired contractile
function of the respiratory muscles, particularly the diaphragm, has
been reported to lead to respiratory failure in experimental studies
(2
, 3)
. Sepsis also contributes to diaphragmatic
impairment in human pathology, particularly in patients with a
critically decreased function of respiratory muscles, such as chronic
obstructive lung disease (4)
, especially during weaning
from mechanical ventilation (5)
.
Recently, we (6)
and others (7
, 8)
have
demonstrated that an excess of nitric oxide
(NO),2synthesized by the inducible isoform of the NO synthase (iNOS), is
involved in septic diaphragm muscle failure. However, these studies did
not examine the effects of NO at the cellular level. NO has been
recognized to affect cellular respiration. Indeed, NO has been
recognized to reversibly inhibit O2 uptake by
binding to cytochrome oxidase in rat skeletal muscle (9)
and heart (10
, 11)
mitochondria, implying a likely
transitory impairment of ATP availability for muscle contraction.
Furthermore, NO reacts with superoxide anion
(O2-·) to form peroxynitrite
(ONOO-) (12)
, which irreversibly
inhibits several mitochondrial enzymes such as aconitase, NADH- and
succinate-dehydrogenases, and superoxide dismutase
(13
14
15)
.
Moreover, we have recently reported that NO inhibits electron transfer
in the mitochondrial respiratory chain at the ubiquinol-cytochrome
b region with a significant increase in the production of
O2-· and hydrogen peroxide
(H2O2) (10
, 11)
, probably by the auto-oxidation of ubisemiquinone
(16)
. Since mitochondria are the most important source of
O2-· in most mammalian organs
and certainly in heart and skeletal muscle (17)
, the large
quantities of NO synthesized by iNOS during sepsis could initiate a
sequence of reactions leading to the formation of
ONOO- and, subsequently, to the nitration of
mitochondrial proteins, resulting in mitochondrial dysfunction.
According to this hypothesis, a recent study by El-Dwairi and
co-workers (8)
shows evidence of
ONOO- formation in the diaphragm of endotoxemic
rats. In that study, however, the potential cellular targets of
peroxynitrite were not examined. Furthermore, to the best of our
knowledge there are no data on diaphragmatic intramitochondrial
formation of ONOO- and its potential toxic
effects on muscular organelles isolated from endotoxemic animals.
Therefore, the aims of this study were to investigate in Escherichia coli endotoxemic rats 1) whether an increased diaphragmatic NO concentration due to iNOS induction resulted in an impairment of mitochondrial energy-linked functions, via generation of ONOO-, and 2) the possible implications of mitochondrial alterations on muscular contractile performance.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The animals were randomly divided into two groups, which received either sterile 0.9% NaCl (control animals, n=58) or 10 mg/kg E. coli endotoxin suspension [lipopolysaccharide (LPS) animals, n=102] intraperitoneally. The endotoxin used was lyophilized E. coli LPS (serotype 0.26 B6; DIFCO, Detroit, Mich.), which was reconstituted with 0.9% sterile sodium chloride.
LPS animals were killed 3 and 6 h after inoculation (LPS-3 and
LPS-6 groups, n=24 and 46, respectively). A third LPS group
received intravenous 8 mg/kg of the NO synthase inhibitor,
NG monomethyl L-arginine (L-NMMA; see
18
for review), 90 min after LPS inoculation (group
LPS-6+L-NMMA, n=32).
C group animals were killed 6 h after inoculation (n=50). A second C group received L-NMMA at the same dose and time as animals from group LPS-6 + L-NMMA (group C+L-NMMA, n=8).
Rats were anesthetized with ether. The diaphragm (with its origins and insertion on the ribs and central tendon left intact) was quickly excised and transferred to a dissecting dish containing oxygenated Krebs solution (composition in mM, NaCl: 137, KCl: 4, MgCl2: 1, KH2PO4: 1, NaHCO3: 12, CaCl2: 2, and glucose 6.5), into which it was pinned to maintain resting length during dissection. Krebs solution was exchanged frequently. Muscular samples from the lateral costal region of each hemidiaphragm were obtained. A set of these samples was carefully prepared to analyze diaphragmatic contractile properties, and another set was freeze-clamped and stored at -80°C until use for detection of iNOS protein and activity.
In a different experimental set, whole diaphragms from animals from groups C, LPS-6, and LPS-6 + L-NMMA were excised to be immediately used to measure diaphragm NO and H2O2 steady-state concentrations. In mitochondrial experiments, the whole costal diaphragm from two rats was used to suspend the organelles at an adequate protein concentration.
Western analysis of iNOS
Frozen diaphragm muscle samples were homogenized with an
Ultraturrax T25 (Janke and Kunkel, IKA Works, Cincinnati, Ohio) in
lysis buffer (Tris HCl 50 mM pH 7.4, EDTA 0.1 mM, leupeptin 1 µM,
PMSF 1 µM, aprotinin 1 µM). The crude homogenates were centrifuged
at 2000 x g for 15 min at 4°C. Proteins of the
supernatant (150 µg per lane) were separated by electrophoresis on
precasted 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel
(Bio-Rad, Richmond, Calif.) and transferred to a PVDF membrane
(Bio-Rad). The membranes were incubated with a rabbit antimurine iNOS
polyclonal antibody (1:250) (Transduction Laboratories, Lexington, Ky.)
raised against a 21 kDa protein fragment corresponding to peptide
sequence 961-1144 of mouse iNOS. The membranes were blotted with a goat
anti-rabbit IgG (1:3000) conjugated to alkaline phosphatase (Bio-Rad),
followed by detection of immunoreactive proteins by a chemiluminescence
method (Bio-Rad). E. coli LPS-stimulated rat alveolar
macrophages obtained by bronchoalveolar lavage were used as a positive
control (6)
.
Diaphragm NOS activity measurements
NOS activity in tissue samples was measured by the conversion of
[3H]-L-arginine to
[3H]-L-Citrulline according to Bredt and Snyder
(19)
. To differentiate iNOS activity, which is independent
of calcium and calmodulin (20)
, and constitutive NOS
(cNOS) isoform activity (calcium and calmodulin dependent), NOS
activity was determined in the presence or absence of 1.2
CaCl2 and 10 µg/ml calmodulin. To calculate the
NOS activity in whole diaphragm, we assumed that proteins account for
~15% of muscle mass (21)
.
Diaphragm NO steady-state concentration
Nitric oxide was electrochemically determined with a Clark-type
amperometric electrode ISO-NOP (World Precision Instruments, Sarasota,
Fla.) in Krebs-Henseleit solution by holding whole diaphragms in a 3 ml
thermostated chamber. Incubation was performed under strong stirring at
30°C. The NO electrode was calibrated daily by a standard chemical
method with known concentrations of NaNO2 in acid
medium (0.1 M H2SO4-KI)
that generates known concentrations of NO. Based on the free diffusion
of nitric oxide through cell membranes, its intracellular steady-state
concentration can be approached by measuring the NO concentration in
the incubation medium, as an expanded extracellular compartment, after
reaching diffusion equilibrium (intracellular concentration equals to
extracellular concentration) (22)
. The NO plateau
concentration was reached after 30–40 min.
Diaphragm H2O2production
Production of H2O2
was continuously monitored with a Hitachi 2000 fluorescence by the
horseradish peroxidase (HRP)/p-hydroxyphenyl acetic acid (p-HPA) assay
(23)
spectrophotometer (Hitachi Ltd., Tokyo, Japan)
supplemented with 12 U/ml HRP and 50 µM p-HPA, with excitation and
emission wavelengths of 315 and 425 nm, respectively. The whole
diaphragm was placed in a thermostated chamber in the experimental
conditions described for NO steady-state measurement. To calculate the
H2O2 production of the
whole tissue, the experiments were performed in duplicate, with or
without 0.1 µM catalase, and aliquots were taken at NO plateau
concentration at 40 min incubation; the difference between both
fluorescence measurements was calculated as
H2O2 concentrations
(24)
.
Isolation of diaphragm mitochondria
Excised diaphragms (mean weight =1 g) were placed in an
ice-cold homogenization medium consisting of 0.23 M mannitol, 70 mM
sucrose, 10 mM Tris-HCl, and 1 mM EDTA with 0.5% bovine albumin (pH
7.4). The tissue was finely minced, transferred to a Teflon, motorized
Potter-Elvejhem homogenizer (Thomas Scientific, Philadelphia, Pa.), and
homogenized in 9 ml cold medium per gram of chopped tissue. The
homogenate was centrifuged at 700 x g at 4°C
for 10 min. The supernatant was decanted and centrifuged at 7000
x g for 10 min. The pellet was washed twice and resuspended
in homogenized medium without bovine albumin with a cooled glass rod up
to a protein concentration of 20 mg/ml (25)
.
Mitochondrial respiratory activities and respiratory control
Mitochondrial oxygen uptake was measured polarographically with
a Clark-type electrode placed in a 3 ml chamber at 30°C in a medium
consisting of 0.23 M mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM
Na2HPO4/KH2PO4,
and 1 mM EDTA (pH 7.4) saturated with room air (0.24 mM) and at 1–2 mg
mitochondrial protein/ml. Oxygen uptake was determined with 6 mM
malate/6 mM glutamate as substrate in the presence (state 3) and
absence (state 4) of phosphate acceptor (0.2 mM ADP). The respiratory
control ratio was calculated as the state 3/state 4 oxygen uptake
ratio. Oxygen uptake was expressed in ngat O/(min/mg prot). The ADP:O
ratio was calculated as the ratio of nanomoles of added ADP divided by
the nanogram atoms of oxygen used during state 3 respiration
(26)
.
Mitochondrial
H2O2 production
The production of
H2O2 was continuously
monitored by the HRP/p-HPA assay. The reaction mixture consisted of
0.23 M mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM
Na2HPO4/KH2PO4,
1 mM EDTA (pH 7.4), 12 U/ml HRP, 50 µM p-HPA, and 0.1–0.5 mg
mitochondrial protein/ml. Succinate (8 mM) was used as substrate and
antimycin was added at 2 nmol/mg prot.
Manganese-superoxide dismutase (Mn-SOD) activity
Mn-SOD was determined by inhibition of the rate of 20 µM
cytochrome c reduction by 0.5 mM xanthine and xanthine
oxidase (initial rate: 0.025 A/min) (27)
. The assay was
carried out in 50 mM potassium phosphate and 0.1 mM EDTA (pH 7.8) at
550 nm. Sodium cyanide (1 mM) was used to inhibit Cu-Zn-SOD and
cytochrome oxidase activities.
Detection of ONOO- generation in mitochondria
This was accomplished by detection of 3-nitrotyrosine residues
in mitochondrial proteins by Western blot with a monoclonal
anti-nitrotyrosine antibody (Upstate Biotechnology Incorporated, Lake
Placid, Mass.) developed by Beckman and co-workers (28)
.
Proteins (60 µg) of the mitochondrial suspension were separated by
electrophoresis on precasted 7.5% SDS-polyacrylamide gel (Bio-Rad,
Richmond, Calif.), transferred to a nitrocellulose membrane (Bio-Rad),
and revealed using the antinitrotyrosine antibody. Bovine serum albumin
(BSA) nitrated after 30 min incubation with 1 mM SIN-1 was used as a
positive control. Incubation of the antibody with 10 mM nitrotyrosine
prior to the membrane incubation was used to ensure the specificity of
the antibody. Red Ponceau staining of crude proteins in the membrane
determined equal loading/transfer among the different lanes.
Protein concentration
Protein concentration was spectrophotometrically measured in
96-well plates using the Bio-Rad protein reagent, with BSA as the
standard.
Diaphragmatic force measurement
Diaphragmatic force was measured as described previously
(6)
. Briefly, bundles from the middle part of the lateral
costal region of the diaphragm (~2 mm wide) were electrically
stimulated by means of rectangular platinum field electrodes. Stimuli
of 0.5 ms were applied using a Grass S 48 Stimulator (Grass
Instruments, Quincy, Mass.) connected in series with a power amplifier
(Grass Instruments). Stimulus intensity was increased until maximal
tension responses were obtained and then set at 1.2-fold the maximal to
ensure supramaximal stimulation (approx. 30 V). Isometric force was
measured with a Grass FT 03 force transducer. Peak twitch force, twitch
kinetics [time to peak twitch tension (TPT), and time to the half of
relaxation (TR)] were measured from a series of contractions induced
by single-pulse stimuli. The force–frequency relation was studied
using 1 s trains of stimuli at 10, 20, 30, 50, 100, and 120 Hz. At
least 1 min intervened between each stimuli train. Force expressed in
Newtons was normalized for muscle cross sectional area, which was
estimated by dividing bundle weight by length and specific density
(1.056 g/cm3) (29)
. Twitch kinetics
was measured as described previously (6)
.
Reagents
SDS, glycerol, 2-(-mercaptoethanol), and bromphenol blue were
obtained from Bio-Rad. [3H]-L-arginine was from
NEN/DuPont (Boston, Mass.). SIN-1 and tetrahydrobiopterin were supplied
by Research Biomedicals Inc. (Natick, Mass.). Rat alveolar macrophage
culture media, supplements, and fetal bovine serum were from Flow Labs
(Irvine, Calif.). Tissue culture plastic ware was supplied by Costar
Corp. (Cambridge, Mass.). Other chemicals were purchased from Sigma
Chemical Company (St. Louis, Mo.).
Statistical analysis
Data were expressed as mean ± SE. To assess
statistical differences, the data were analyzed by one-way analysis of
variance and the Dunnet's test. Statistical significance was accepted
when P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A). The iNOS protein was detected 3 and 6 h after LPS
inoculation. The iNOS activity paralleled iNOS protein expression,
being higher in the LPS-3 and LPS-6 samples than in C samples
(P<0.01, Fig. 1B
); this increase in iNOS
activity was prevented by L-NMMA administration. No differences in iNOS
activity were assessed between LPS-3 and LPS-6 samples.
|
Constitutive NOS activity was similar in diaphragm homogenates from
groups C, LPS-3, and LPS-6 (Fig. 1B
). By contrast, this
activity was significantly lower in C+L-NMMA than in C samples and in
LPS-6 + L-NMMA than in LPS-6 samples (P<0.05 in each case;
Fig. 1B
).
NO steady-state concentration
The diaphragm NO steady-state concentration, determined at
diffusion equilibrium in an expanded extracellular medium, was 0.5 ± 0.01 µM NO and 0.18 ± 0.01 µM NO in the muscles from LPS-6
and LPS-6 + L-NMMA injected rats, respectively, whereas NO steady-state
concentration in nonstimulated diaphragm was almost 0.02 µM. For
comparison, a steady-state concentration of 0.06 ± 0.002 µM NO
was measured in diaphragms stimulated with bradykinin, an activator of
constitutive NOS (Fig. 2
). The increased NO steady-state concentration observed in LPS-6 animals
was suppressed by addition of L-NMMA to the media (data not shown).
|
Diaphragm H2O2 production
The levels of H2O2
detected in the incubation media from diaphragms of LPS-6 animals were
significantly higher than from C animals (P<0.05, Fig. 2
,
inset). This increase was suppressed either by addition of L-NMMA to
the media (data not shown) or by previous L-NMMA administration;
H2O2 production was
significantly lower in LPS-6 + L-NMMA than in LPS-6 animals
(P<0.05), not different from C animals (Fig. 2
, inset).
Mitochondrial oxygen uptake
Mitochondria isolated from LPS-treated rats showed a 7 and 33%
decrease in respiratory control 3 h and 6 h after LPS
inoculation, respectively (Table 1
). These data are consistent with the partial uncoupling of oxidative
phosphorylation to electron transfer. Accordingly, the ADP:O ratio was
reduced by 33 and 39% in the same organelles. Administration of L-NMMA
prevented partially the impairment of mitochondrial function in
LPS-6-injected rats. Administration of L-NMMA to control animals only
slightly increased oxygen uptake rates (by ~10%), but did not modify
mitochondrial respiratory control or ADP:O ratios (Table 1)
.
|
Mitochondrial hydrogen peroxide production
Mitochondria from endotoxemic animals supplemented with antimycin
showed an increased production rate of
H2O2 reflecting a higher
intramitochondrial concentration of nondiffusible superoxide radicals,
the products of the univalent reduction of oxygen. The mitochondrial
production rate of H2O2 was
increased by 84 and 176%, with respect to control organelles, in
samples from groups LPS-3 and LPS-6, respectively (P<0.05,
Table 1
). The increased rate of
H2O2 production in
antimycin-supplemented mitochondria is consistent with partially
uncoupled mitochondria. Treatment of the animals with L-NMMA, resulted
in a partial prevention of the increase in mitochondrial
H2O2 production rate (Table 1)
. Administration of L-NMMA to control animals did not modify
mitochondrial H2O2
production rate (Table 1)
.
NO-induced hydrogen peroxide production by diaphragm mitochondria
Rat diaphragm mitochondria supplemented with NO showed a
dose-dependent H2O2
production; the maximal
H2O2 production rate was
achieved at ~0.25 µM NO (Fig. 3
) and accounted for ~50–65% of the rate obtained in the presence of
antimycin.
|
Mn-SOD content in diaphragm mitochondria
To estimate the intramitochondrial steady-state concentration of
superoxide anion, we measured Mn-SOD concentration in mitochondrial
homogenates. The content of mitochondrial Mn-SOD (µM) was not
statistically different among the groups (1.9 ±0.2control
group, 2.2 ±0.3 in both LPS-3 and LPS-6 groups and 1.9 ±0.2
in LPS-6+L-NMMA group, n=8 for each group, respectively).
Nitrotyrosine residues in mitochondria
Western blot analysis of mitochondrial homogenates using a
monoclonal antinitrotyrosine antibody showed that endotoxemic animals
exhibit a reproducible pattern of protein nitration for several bands
(Fig. 4
A). Immunoblot intensity was higher in LPS-6 than in LPS-3
samples, which showed only bands of very weak intensity. Mitochondrial
protein nitration during endotoxemia was similar to the one observed
after 30 min exposure of mitochondria to 1 mM SIN-1 (Fig. 4A
). Nitrated protein bands were markedly decreased by the
previous administration of L-NMMA to the animals (Fig. 4B
).
The preincubation of the antinitrotyrosine antibody with free
nitrotyrosine significantly attenuated the blotting signal, thus
ensuring the specificity of the antibody (data not shown). Red Ponceau
staining was similar in all lanes (data not shown).
|
Diaphragmatic contraction force
Diaphragmatic force was significantly reduced in LPS-6 but not in
LPS-3 animals. At that time, peak twitch and maximal tetanic tension
were both reduced by ~50% (Fig. 5
). Analysis of diaphragmatic force–frequency relationships showed that
the contraction force generated in response to all stimulation
frequencies was lower in LPS-6 than in LPS-3 and control muscles. TPT
and TR were similar in LPS and control animals. The reduction of
diaphragmatic force observed in LPS-6 animals was significantly
attenuated by administration of L-NMMA (Fig. 5)
. Administration of
L-NMMA to control animals did not modify diaphragmatic force generation
capacity (Fig. 5)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The uncoupling of oxidative phosphorylation and the increase in
H2O2 production rate
observed in endotoxemic animals are probably related to multiple
effects of E. coli endotoxin inoculation upon mitochondrial
electron transfer and ATP synthesis. One of these effects should be an
inhibition of the electron transfer at the ubiquinone-cytochrome
b region, which is required to elicit a significant
production of O2–· and its
diffusible dismutation product,
H2O2 (10
, 16)
.
These results agree with previous reports that proposed oxidative
stress as a mechanism of diaphragmatic dysfunction during endotoxemia
(30
, 31
, 32)
. In those studies, oxidative stress was
assessed indirectly on the basis of an increase in muscular lipid
peroxidation end products like malondialdehyde or 8-isoprostane. A
direct measurement of H2O2
released by mitochondria and by the whole diaphragm as presented here
is evidence of the presence of oxidative stress and indicates for the
first time that mitochondria are the main source of the excess of
oxygen free species.
Mitochondrial alterations may be mediated by different mediators of the
inflammatory cascade; for example, tumor necrosis factor 
has been
reported to uncouple oxidative phosphorylation by enhancing the
mitochondrial production of oxygen free radicals (33)
. In
the present study, the increase in
H2O2 released by the
diaphragm of LPS-6 animals was in parallel with the marked increase in
the NO released by the muscle of these animals, suggesting a connection
between diaphragmatic oxidative stress and NO tissue levels. This was
further confirmed by the significant attenuation of both mitochondrial
H2O2 production rate and
diaphragm H2O2 detection
observed after L-NMMA administration to animals of the LPS-6 group.
This contributive role of NO to oxidative stress in endotoxemic animals
is likely to represent the direct effects of NO, which increased
H2O2 release in normal
diaphragm mitochondria (Fig. 3)
. The NO-dependent production of
O2–· and
H2O2 (NO
O2–·
H2O2) in diaphragm
mitochondria agrees with similar findings in rat heart mitochondria
(10)
and in isolated beating rat heart (11)
.
Recently, we have observed that NO-induced
O2–· production could be
sustained by a direct reaction of NO with ubiquinol through the
formation of the auto-oxidable intermediate semiquinone.
Although in this model iNOS appeared to be the primary NO source in the
diaphragm of endotoxemic animals, we could also discuss a possible role
of muscle cNOS. In accord, Kobzik and colleagues (34a)
documented for the first time the presence of the neuronal NOS (nNOS)
located beneath the sarcolemma of fast-twitch normal muscle fibers and
demonstrated that NO depressed contractile force generation. The same
group (35)
reported the presence of endothelial NOS (eNOS)
in the mitochondrial fraction of skeletal muscle whereas other studies
have confirmed the existence of eNOS in mitochondria from cardiac
myocytes, hepatocytes, and kidney cells (36)
. Furthermore,
a specific mitochondrial NOS has been recently described in rat
hepatocytes (37)
. The NO synthesized by this isoform
exerts a negative influence on normal mitochondrial respiration
(38)
. Therefore, if NO synthesized by both cNOS may have
played a similar role in control and endotoxemic animals, the
protective effects of L-NMMA in LPS-6 animals could be interpreted as
an extrapolation of its effects in control animals and not as a
specific effect in endotoxemia. However, to our knowledge all described
constitutive and mitochondrial NOS have, irrespective of their
primary structure, a calcium–calmodulin-dependent activity,
which was unchanged in this study. Moreover, at the administered
L-NMMA dose, we did not observe significant modifications of
mitochondrial respiratory control index or diaphragm contractile force
in the samples from control animals, in agreement with data from
previous studies. In accord, Kobzic and co-workers (34a)
reported only a modest increase (~15%) in isometric force after the
incubation of diaphragmatic bundles with a very high concentration of
the NOS inhibitor nitro-L-arginine (LNA, 10 mM). Even neglecting this
very high LNA concentration, it is evident that a potential 15% change
in contractile force in animals from group C does not account for the
50% increase in muscle force induced by L-NMMA in animals from group
LPS-6. On this basis and regardless of the specific significance of
both cNOS isoforms in diaphragm muscle, it is likely that in the
present experimental model, most preventive effects of L-NMMA are due
to the inhibition of iNOS, though some additional effects on cytosolic
or mitochondrial constitutive NOS cannot be completely ruled out.
Considering that the reaction of NO with
O2–· to yield
ONOO- is a diffusion-controlled reaction
(39)
, a simultaneous increase in intramitochondrial
O2–· and NO steady-state
concentrations will increase the rate of ONOO-
formation. Peroxynitrite should be the ultimate mediator of the
described mitochondrial and cellular impairment. Accordingly, isolated
diaphragm mitochondria did not show a marked inhibition of oxygen
uptake rate, consistent with a reversible NO inhibition of cytochrome
oxidase (9
, 11)
, but did show uncoupling and sustained
O2–· production consistent
with irreversible effects of ONOO-
(14)
.
According to the steady-state approach, the rate of
O2–· production
(d[O2·]/dt)
equals the rate of O2–·
utilization
(-d[O2·]/dt);
considering H2O2 and
ONOO- as the two products of
O2–· utilization:
 |
),
the stoichiometric relationship of 2
O2–· per
H2O2 (17)
and
50 mg of mitochondrial proteins per gram of muscle (40)
,
the intramitochondrial concentration of
O2–· should be:
 |
). These data support the idea that during
endotoxemia, both H2O2 and
peroxynitrite are generated in vivo in diaphragm
mitochondria. In fact, detection of nitrated tyrosine residues in
mitochondrial proteins adds strong evidence about a previous exposure
of mitochondrial proteins to endogenous peroxynitrite. This agrees with
recent in vitro data published by Szabo and co-workers
(41)
in murine macrophages, stimulated with LPS and
interferon-
showing oxidation of mitochondrial proteins that was
inhibited by L-NMMA. The two major nitrated bands in mitochondrial
samples from LPS-6 animals (106 and 69 kDa, respectively) are similar
to two bands recently described by Cuzzocrea and associates
(42)
in homogenates of macrophages from rats subjected to
carrageenan-induced pleurisy. In these experiments, the authors blotted
all cellular proteins, including the mitochondrial fraction. As in this
study, the bands were greatly attenuated by pretreating the cells with
an NOS inhibitor.
Thus, time course differences in the coupling of mitochondria isolated
from LPS-inoculated rats (Table 1)
can be attributed to progressive
effects of peroxynitrite. Nitration of proteins at their tyrosine
residues should linearly depend on the steady-state concentration and
on the time of exposure to ONOO-. Most
ONOO- effects on mitochondria and cells were
assessed after the addition of a single pulse of
ONOO-, which rapidly decays to nitrate
(k =0.64 s-1 at pH 7.0 and
37°C) (43)
. For fast-decaying molecules, the chemical
effects can be analyzed in terms of the total amount of molecules added
to the system. Therefore, we are able to compare the exposure to
peroxynitrite when ONOO- is added as a pulse or
continuously generated. Peroxynitrite toxicity was, for example,
assayed adding a pulse of 750 µM ONOO- to kill
E. coli (43)
. In the present study, we
considered a constant ONOO- production during
3 h of endotoxemia, and exposed mitochondria to ~600 µM
ONOO- (0.6 x10-7 M
s-1x60 sx180 min, Fig. 4B
), a
concentration and number of molecular collisions similar to that which
killed E. coli. Albumin nitration by SIN-1 (Fig. 4)
occurred
at an ONOO- production rate of 10 µM/min after
30 min, corresponding to an exposure to 300 µM
ONOO-. This comparison adds evidence that
extensive nitration of mitochondrial proteins in endotoxemia occurs
in vivo and that a critical NO and
ONOO- exposure time must be reached until the
deleterious effects become apparent.
Nitration of mitochondrial proteins and maximal impairment of
mitochondrial function were associated with a significant decrease in
diaphragmatic force generation. In the presence of less important
mitochondrial alterations and with already increased iNOS activity (3 h
after LPS), diaphragmatic force remained unchanged. A similar temporal
relationship was described by Xie and co-workers (44)
where the exposure of rat cardiac muscle to a continuous flow of
ONOO- induced a parallel and irreversible
30–40% decay in myocardial O2 uptake and
contractile amplitude, whereas more modest and reversible effects were
observed with NO released by the NO donor SNAP. These elements suggest
that despite the complexity of the involved mechanisms, an irreversible
ONOO--mediated diaphragmatic mitochondrial
dysfunction and contractile failure were probably causally related.
Indeed, muscular contractile performance has been shown to be impaired
by uncouplers and inhibitors of the mitochondrial electron transfer
chain like dinitrophenol (45)
or diphenyleneiodonium
(46)
. In addition, on the basis of recent studies
(47
, 48)
, mitochondrial dysfunction could lead to
contractile impairment via other mechanisms, like apoptosis
of skeletal myocytes. Finally, it must be noted that
ONOO- could impair diaphragmatic force by
extramitochondrial mechanisms. Indeed, ONOO- can
affect calcium homeostasis by inactivating
Ca2+-ATPase (49)
and can modify
actin properties (50)
. However, since extramitochondrial
synthesis of O2–· is probably
very low, only a sustained diffusion of the short-life
ONOO- outside the mitochondria could affect
these or other extramitochondrial proteins involved in
excitation-contraction coupling. In this context, it is interesting to
note a recent study by El-Dwairi and colleagues (8)
reporting the existence of two faint bands of 42 and 50 kDa proteins
blotted with the antinitrotyrosine antibody in the cytosolic fraction
of whole diaphragm homogenates from control and endotoxemic rats, and
two bands of 86 and 196 kDa, respectively, only in endotoxemic animals
(8)
. However, the molecular mass of the two bands found in
endotoxemic rats is different from that of the potentially nitrable
Ca2+-ATPase and actin proteins (110 and 60 kDa,
respectively; ref 51
). Moreover, lack of modifications of twitch
kinetics in endotoxemic animals does not support the alterations of
these proteins, especially Ca2+-ATPase
(52)
. Nevertheless, we cannot definitively exclude the
possibility that extramitochondrial alterations participate in the
genesis of diaphragmatic force impairment in endotoxemic animals.
In a previous study, we suggested that the production of
O2–· in mitochondria may be a
physiological oxidative NO detoxifying pathway (10)
. The
results of the present study show that amplification of this pathway
may transform it into a dangerous pathogenic mechanism, leading to
mitochondria and, probably, organ failure. On this basis, oxidative
stress in the diaphragm during sepsis could reflect NO overproduction
by iNOS.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication September 30, 1998. Revised for publication March 12, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. S. Supinski and L. A. Callahan Free radical-mediated skeletal muscle dysfunction in inflammatory conditions J Appl Physiol, May 1, 2007; 102(5): 2056 - 2063. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Carreras and J. J. Poderoso Mitochondrial nitric oxide in the signaling of cell integrated responses Am J Physiol Cell Physiol, May 1, 2007; 292(5): C1569 - C1580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Vassilakopoulos, K. Govindaraju, D. Parthenis, D. H. Eidelman, Y. Watanabe, and S. N. A. Hussain Nitric oxide production in the ventilatory muscles in response to acute resistive loading Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L1013 - L1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Callahan and G. S. Supinski Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis J Appl Physiol, January 1, 2007; 102(1): 44 - 53. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Fredriksson, F. Hammarqvist, K. Strigard, K. Hultenby, O. Ljungqvist, J. Wernerman, and O. Rooyackers Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E1044 - E1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Converso, C. Taille, M. C. Carreras, A. Jaitovich, J. J. Poderoso, and J. Boczkowski HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism FASEB J, June 1, 2006; 20(8): 1236 - 1238. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Supinski and L. A. Callahan Polyethylene Glycol-Superoxide Dismutase Prevents Endotoxin-induced Cardiac Dysfunction Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1240 - 1247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Callahan and G. S. Supinski Sepsis Induces Diaphragm Electron Transport Chain Dysfunction and Protein Depletion Am. J. Respir. Crit. Care Med., October 1, 2005; 172(7): 861 - 868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
