(Circulation. 1997;96:614-620.)
© 1997 American Heart Association, Inc.
Articles |
Lactate and PO2 Modulate Superoxide Anion Production in Bovine Cardiac Myocytes
Potential Role of NADH Oxidase
From the Department of Physiology, New York Medical College, Valhalla, NY.
Correspondence to Michael S. Wolin, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background Lactate increases lucigenin chemiluminescence (CL)–detectable superoxide anion (O2
-) generation in bovine vascular smooth
muscle and endothelium, and a microsomal
flavoprotein-containing NADH oxidase whose activity is regulated by
PO2 and cytosolic NAD(H) redox appears to be
the detected source of O2-
production. Little is known about the importance of this
O2--producing system in cardiac
myocytes.
Methods and Results In isolated bovine cardiac myocytes,
lactate (10 mmol/L) increased lucigenin-detectable
O2- levels to 
1.8 times baseline,
whereas pyruvate (10 mmol/L) and mitochondrial probes did not
increase the detection of O2-. A
nonmitochondrial NADH oxidase activity, found in microsomes containing
a cytochrome b558, was a major source of
O2- production in the
homogenate of myocytes, because NADH (0.1 mmol/L)
increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria,
and xanthine oxidase were minor sources of detectable
O2- production. However,
mitochondria released H2O2 in the presence of
5 mmol/L succinate and 30 µmol/L antimycin, based on its
detection as catalase-inhibitable luminol (+horseradish
peroxidase)–elicited CL. Diphenyliodonium (DPI), an
inhibitor of flavoprotein-containing oxidases,
significantly attenuated basal, lactate, and NADH-elicited lucigenin
CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic
reoxygenation caused an 8.6-fold increase in the
detection of O2- that was potentiated by
lactate and inhibited by DPI.
Conclusions NADH oxidase activity linked to cytosolic
NAD(H) redox appears to be a key source of
O2- production in cardiac myocytes
that could contribute to oxidant signaling mechanisms and injury upon
exposure to changes in PO2 and metabolites
produced under hypoxia, such as lactate. These processes could
contribute to the previously observed potentiation of injury caused by
lactate in cardiac ischemia/reperfusion.
Key Words: free radicals • hypoxia • myocytes • oxygen
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Reactive oxygen species seem to play an important role in the pathophysiology of H/R and I/R injury in various organs, including the myocardium.1 2 3 4 Although examination of the significance of ROS in injury to cardiac muscle has been a major area of investigation, the importance of certain potential sources of production of these species has not been systematically studied. Mitochondria and xanthine oxidase have often been implicated in this increase in ROS after H/R or I/R in myocardium.1 2 3 4 5 6 7 Our group has previously investigated in isolated bovine microvessels and myocytes the effect of a 30-minute hypoxia followed by reoxygenation treatment on the steady-state level of an endogenous basal source of O2
- that was detected by
lucigenin-elicited CL.7 In this previous study, the basal
level of O2- was not altered by H/R in
either the microvessels or myocytes. However, the addition of substrate
for xanthine oxidase caused an increase in lucigenin-detectable
O2- in microvessels but not myocytes
exposed to H/R, consistent with an increased expression of
xanthine oxidase activity in the microvessels. In general, only minimal
consideration has been given to the possibility that there might be
sources of ROS generation in cardiac muscle other than xanthine oxidase
and mitochondria.
We have previously reported evidence in isolated
endothelium-removed bovine calf pulmonary and
coronary artery and coronary artery
endothelial cells that O2-
production by an NADH-oxidoreductase(s) with NADH oxidase
activity is the major source of O2-
detected in these tissues by lucigenin-elicited CL.8 9 10 In
these tissues, NADH oxidase activity seems to be linked to the redox
state of cytosolic NADH, because lactate but not pyruvate increases the
observed level of O2-.10 11 12 13
Previous studies examining the role of NADH oxidase as a source of
production of O2- (or
H2O2) in cardiac muscle have focused on
developing the concept that it originates from NADH dehydrogenase of
the mitochondrial electron transport chain14 15 or a
mitochondrial system that has been claimed to have the rather unusual
property of being able to directly utilize extramitochondrial
NADH.16 17 The O2--producing
oxidase activity our group has characterized in vascular tissue
utilizes predominantly NADH, not NADPH, and its rate of
O2- production is sensitive to
physiological changes in
PO2. Thus, the requirements of this system for
NADH and oxygen as substrates permit it to potentially function as a
cellular sensor of metabolic states that alter cytosolic
NAD(H) redox and PO2.9 11 12 13 18 19
Our previous studies in both pulmonary and coronary
arterial smooth muscle suggest that many of the responses
of these vascular segments to changes in PO2
are activated as a result of ROS produced by this oxidase, and
in both of these vascular segments, posthypoxic
reoxygenation is associated with a transient
overproduction of
O2-.9 19 We have also shown
that NADH-dependent O2- production
appeared to originate from a microsomal electron transport chain
containing a DPI-inhibitable flavoprotein site and spectrally
detectable cytochrome b558.18 19
The objective of the present study was to examine whether an NADH
oxidase linked to cytosolic NAD(H) redox was an important source of
O2- production in isolated and
homogenized calf cardiac myocytes.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Lucigenin (bis-N-methylacridinium nitrate), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), HEPES, MOPS, NADH, NAD, NADPH, lactic acid, sodium pyruvate, sodium succinate, antimycin A, rotenone, sodium arachidonate, hypoxanthine, catalase (bovine liver), horseradish peroxidase (type VI), SOD (bovine blood), and Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid) were purchased from Sigma Chemical Co. DPI chloride was purchased from Aldrich Chemical Co. Other chemicals were analyzed reagent grade from Baker Chemical Co. Lactate solutions (1 mol/L) were prepared by dissolving lactic acid in water followed by adjustment of pH with NaOH (1 mol/L) to
7.4.
Preparation of Calf Heart Myocytes
Calf heart myocytes were prepared as previously
described.7 Ventricular muscle obtained from
slaughterhouse-derived bovine calf hearts was finely ground with a
commercial meat processor at 4°C. The resulting myocardial tissue was
suspended in a 1:3 dilution of Krebs-HEPES buffer at 4°C containing
the following (mmol/L): NaCl 118, KCl 4.7, CaCl2 1.5,
MgSO4 1.1, KH2PO4 1.2, glucose 5.6,
and HEPES 10, adjusted to pH 7.4 with NaOH. The muscle suspension was
then homogenized at the maximum speed for five periods of 5
seconds in a Sorvall Omnimixer (Du Pont Instruments, Sorvall Biomedical
Division). The homogenized tissue was poured over a 95-µm
nylon mesh sieve (Small Parts Inc) to remove the microvessel fraction
and other larger tissue components. The brownish filtrate containing
cardiac myocytes was then examined by light microscopy. If the
preparation appeared to contain <90% cardiac myocytes and/or if
fibrous connective tissue, microvessel segments, or nonmyocyte
tissue components were observed, the myocyte fraction was briefly
rehomogenized and sieved through 75-µm mesh. The final
filtrate was centrifuged at 650g (4°C) for
separation of intact myocytes from broken myocytes and other tissue
elements. The resulting pellet was then extensively washed with cooled
Krebs-HEPES solution over 25-µm mesh. When examined by light contrast
microscopy, the myocyte fraction obtained from this procedure contained
>90% cardiac myocytes, generally in the form of rod-shaped single
myofibrils or as cells aligned in short chains or small clusters free
of detectable vascular elements. Isolated myocytes were then incubated
for 30 to 45 minutes in Krebs bicarbonate (containing 25 mmol/L
NaHCO3) in a tissue bath gassed with 21% air/5%
CO2 before their immediate use in the experiments
described. When intact myocytes were studied, their viability was
assessed as previously described.7
Preparation of the Homogenate Fraction and Microsomes
From Calf Heart Myocytes
The calf heart myocyte homogenate was prepared by
adaptation of methods previously used with vascular myocytes and
endothelium.9 10 11 Briefly, after isolation,
myocytes (1 g blotted wet wt/30 mL buffer) were homogenized
in 20 mmol/L MOPS-KOH buffer (pH 7.4) containing 250 mmol/L
sucrose at 0°C to 5°C in an Eberbach homogenizer at
maximum speed with five treatments of 20-second duration. A microsomal
fraction was prepared from the homogenate by differential
centrifugation by adapting methods previously used with
vascular myocytes.20 Briefly, the homogenate
was centrifuged at 29 000g for 15 minutes, and the
supernatant obtained was centrifuged at 100 000g
for 1 hour. The pelleted material was resuspended in MOPS-sucrose
buffer for studies on cardiac myocyte microsomes, and the
post–100 000g supernatant fraction was saved for use as a
control in studies on microsomes.
CL Measurement of O2- and
H2O2 Production
By methods previously described,7 intact calf heart
myocytes were placed in plastic scintillation minivials containing
250 µmol/L lucigenin and other additions in a final volume of 1
mL air-equilibrated Krebs solution buffered with 10 mmol/L
HEPES-NaOH (pH 7.4). Experiments on the homogenate fraction
used 0.05 to 0.1 mg/mL protein and were conducted in air-equilibrated
MOPS-sucrose buffer, pH 7.4, containing 50 µmol/L
lucigenin.8 9 10 The CL elicited by
O2- in the presence of lucigenin was
measured in a liquid scintillation counter (Mark V, TmAnalytic) with a
single active photomultiplier tube positioned in out-of-coincidence
mode. All manipulations were performed in the darkroom with minimal
light. Samples were initially at 37°C, but the temperatures
subsequently equilibrated with the ambient temperature of 34°C.
After a 5-minute dark adaptation, vials containing all components, with
the exception of intact myocytes (blanks), were counted once for 0.1
minute and then recounted three times after myocytes (150 mg) were
placed in each vial. Blanks of 103 counts were then
subtracted from the average of the relatively constant levels of CL
produced under each condition by the myocytes to obtain the data
reported as cpm/g myocytes in the "Results." In studies on tissue
homogenate samples, after quantification of the vial blank,
samples were counted immediately within 2 minutes of addition of
substrates for O2- production to
the myocyte homogenate fraction to minimize the effects of
time-dependent changes in metabolite levels (eg, NADH). The sensitivity
and chemical specificity of the lucigenin-elicited light-yielding
reaction for O2- has been reported
previously.21 For peroxide detection, 10 µmol/L
luminol plus 1 µmol/L horseradish peroxidase were substituted
for lucigenin.22
Experiments in which intact myocytes were studied for the effects of
changes in PO2 on CL measurement of
O2- were conducted in a single photon
counting apparatus constructed in a light-tight box similar
to that previously described.9 19 In these experiments,
intact myocytes (0.8 to 1.0 g/mL) were incubated in Krebs
bicarbonate buffer containing 0.25 mmol/L lucigenin in a
continuously gassed 1-cm2 spectrophotometer cuvette mounted
in a thermostatted (37°C) cell holder on the surface of a Lucite
light guide (with a shutter cover) directed into a cooled Thorn EMI
photomultiplier tube (model 9235B). A Thorn EMI amplifier-discriminator
(model C604) and photon counter (model C660) were used to quantify CL.
The counts were integrated over 5-second periods by the photon counter,
and an analog signal of the integrated counts was continuously
recorded on a Grass model 7 polygraph recorder. Data are
reported as counts·5 s-1·g
myocytes-1 after subtraction of the background
of 250 counts/5 s.
Spectral Detection of Microsomal Cytochromes
Spectral studies were conducted as previously
described18 in 50 mmol/L sodium phosphate buffer, pH
7.4, with 0.5 mg/mL microsomal protein in both the sample and
reference cuvettes. After the baseline or oxidized cytochrome spectra
were re-corded over the 500- to 620-nm range, a few grains (to
avoid sample dilution) of sodium dithionite were added to the sample
cuvette to generate reduced cytochromes in an anaerobic
environment, and these spectra were subsequently recorded
immediately after dithionite was added.
Statistical Analysis
Data were evaluated by a one-way ANOVA with Scheffé's
post hoc test to determine statistical significance between groups. The
acceptable level of significance was P<.05. The number of
experimental determinations (n) in all cases is equal to the number of
animals from which myocytes were obtained. Data in the figures are
depicted as mean±SEM.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of Lactate, Mitochondrial Inhibitors, and DPI on Lucigenin-Detectable O2
- in Isolated
Calf Heart MyocytesExperiments in our previous study on isolated bovine cardiac myocytes using lucigenin for the detection of O2
- indicated that inhibition of Cu,Zn-SOD
increased the level of CL measured by the scintillation counter
method.7 In that previous study, it was also observed that
the tissue-permeable scavenger of O2- Tiron
but not added SOD decreased the amount of CL detected. Thus, basal
levels of myocyte-derived lucigenin CL appear to originate from an
intracellular source of O2-
production. As shown by the data in Fig 1
, 10
mmol/L lactate increased the amount of CL detected to 1.8
times baseline. To evaluate whether mitochondrial NADH oxidase
activity14 15 participates in the sources of
O2- detected by lucigenin, the effects of
two inhibitors of mitochondrial electron transport,
rotenone and antimycin, were evaluated. As shown by the data in Fig 1, these inhibitors of mitochondrial electron transport did
not significantly alter the amount of basal or lactate-elicited CL
detected. The mitochondrial probes may actually have a small
stimulating effect on basal CL, because the increase in
lactate-elicited CL did not reach statistical significance in the
presence of these probes. As shown in Fig 1, the flavoprotein
inhibitor DPI (10 µmol/L) reduced basal lucigenin CL
by 27%, and this probe completely eliminated the lactate-elicited
increase in the detection of O2-.
|
Conditions for the Detection of Lactate-Stimulated
O2- Production in
Homogenized Calf Heart Myocytes
In the homogenate fraction of calf heart
myocytes, lactate was able to increase CL only in the presence of added
NAD (Fig 2A), presumably as a result of NADH formation
by lactate dehydrogenase. As shown by the data in Fig 2A, lactate,
pyruvate, NAD, or pyruvate+NAD did not elicit detectable
increases in CL over the background level of the myocyte
homogenate. As shown by the data in Fig 2B, NADH (0.1
mmol/L), a product of the lactate dehydrogenase reaction, caused a
prominent increase in lucigenin CL. The increase in CL caused by NADH
was significantly decreased by the scavengers of
O2- SOD (3 µmol/L) and Tiron
(10 mmol/L), further implicating O2-
as the source of CL. Preincubation of the homogenate of
calf heart myocytes for 15 minutes with the flavoprotein
inhibitor DPI (1 µmol/L) also resulted in a
significant decrease in the observed level of NADH-derived CL (Fig 2B).
|
Evidence That NADH Oxidase Is a Major Source of
Lucigenin-Detectable O2- Production
in the Homogenate of Calf Heart Myocytes
Like our studies in calf pulmonary and coronary
arteries,8 9 experiments were conducted with lucigenin for
the detection of sources of O2-
production potentially present in the
homogenate of calf heart myocytes.
O2- production from xanthine
oxidase was determined by examining the increase in lucigenin-elicited
CL caused by 100 µmol/L hypoxanthine. NAD(P)H oxidoreductase was
examined by determining the effect of 100 µmol/L NADH or NADPH,
and arachidonic acid–metabolizing enzymes were
determined by quantifying the increase in CL produced by 10
µmol/L arachidonic acid over the levels observed in
the presence of NADPH. The data in Fig 3A indicate that
NADH oxidase activity was the major source of
O2- production detected by
lucigenin. Xanthine oxidase, arachidonic
acid–metabolizing enzymes, and NADPH oxidases were observed to be
minor sources of O2- production by
this method. Therefore, NADH oxidase appears to be a major source of
O2- detected by lucigenin in the
homogenate of calf heart myocytes.
|
Mitochondria as a Source of O2-
Production in Calf Heart Myocytes
Because lucigenin is a poor detector of mitochondrial electron
transport chain–derived O2-
production,8 we examined the intramitochondrial
production of O2- indirectly by
measuring catalase-inhibitable luminol (10 µmol/L)+horseradish
peroxidase (1 µmol/L)–dependent CL for detection of
H2O2 released from mitochondria in the presence
of combinations of 5 mmol/L succinate and 30 µmol/L
antimycin. Mitochondrial O2-
production is known to be maximized by the presence of the
combination of these two agents, and the H2O2
released is thought to originate from intramitochondrial sources of
O2- production.15 As
shown in Fig 3B
, the combination of succinate and antimycin caused the
largest observed increase in luminol CL, and this increase was
inhibited back to basal levels by 1 µmol/L catalase. Under
similar conditions, lucigenin did not detect an increase in
O2- in the presence of succinate and
antimycin (see Fig 3A), which is consistent with lucigenin
being a poor detector of mitochondrial O2-
production. The increase in lucigenin-detectable
O2- production produced by NADH
(5.56±0.55x107 cpm/mg, n=9) in the myocyte
homogenate also appeared to be independent of mitochondrial
function, because its rate of O2-
production was not significantly altered by the presence of 30
µmol/L rotenone (5.11±0.57x107 cpm/mg, n=9).
Detection of a Cytochrome b558 in
Microsomes Containing NADH Oxidase Activity
In preliminary experiments, conducted in a manner similar to our
studies on NADH oxidase in vascular tissue,20 it was
observed that microsomal membranes obtained by differential
centrifugation of the homogenate contained
the NADH-dependent O2--producing activity.
In addition, an NADH-dependent SOD-inhibitable source of lucigenin CL
could not be detected in the supernatant fraction obtained after
removal of the microsomes by centrifugation at
100 000g. When this microsomal fraction was examined for
the presence of a cytochrome b558 by spectral
methods under anoxic reducing conditions produced by dithionite, a
prominent absorbance was observed at 558 nm (see Fig 4).
|
Effects of H/R in the Absence and Presence of Lactate, Pyruvate, or
DPI on the Detection of O2- by Lucigenin in
Isolated Calf Heart Myocytes
Time-dependent changes in lucigenin CL from isolated calf heart
myocytes as a function of exposure to H/R were measured in the single
photon counting CL detection apparatus described in
"Methods." Fig 5 contains summary data showing the
steady-state level of CL above the lucigenin-containing buffer
background elicited by the presence of myocytes under an atmosphere of
21% O2/5% CO2 (balance N2;
PO2150 mm Hg). This basal level of
production of O2- by myocytes was
essentially eliminated upon exposure to a hypoxic environment
(PO28 to 10 mm Hg) produced by a
subsequent gassing of the myocytes with 95% N2/5%
CO2. As shown in Fig 5, reoxygenation with
21% O2 after a 30-minute exposure to hypoxia
produced a transient 8.6-fold increase in CL over the initial levels
observed with 21% O2, followed by a subsequent decay over
20 minutes back to the levels initially observed under 21%
O2.
|
In intact calf heart myocytes, 10 mmol/L lactate produced a
significant increase in the steady-state level of myocyte lucigenin CL
observed under 21% O2 atmosphere, whereas the presence of
10 mmol/L pyruvate or 10 µmol/L DPI depressed the detected
level of O2- (see Fig 5). None of these
probes altered the essentially undetectable level of CL observed under
hypoxia. The presence of lactate but not pyruvate caused a
marked potentiation of detection of the transient increase in lucigenin
CL observed during posthypoxic reoxygenation. The
presence of DPI markedly reduced but did not eliminate the transient
increase in the level of lucigenin CL observed during posthypoxic
reoxygenation.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results of the present study are consistent with bovine calf cardiac myocytes having a lucigenin-detectable basal source of O2
- production that is increased
by lactate. This basal source of O2-
production in intact myocytes was also shown to be regulated by
changes in PO2 and to be dependent on a
flavoprotein-containing oxidase. The major source of
O2- production detected by
lucigenin in the homogenate of calf heart myocytes is
derived from flavoprotein-dependent NADH oxidase activity, which
appears to originate from a microsomal electron transport chain
containing a cytochrome b558. As might be
anticipated, O2--producing systems
associated with other tissues, such as xanthine oxidase and NADPH
oxidases or arachidonic acid–metabolizing enzymes,
were not major sources of O2- generation in
calf heart myocytes. Although mitochondria seem to be a significant
site of generation of H2O2, lucigenin does not
appear to detect the intramitochondrial pool of
O2- that is likely to be the source of
H2O2 observed to be released from mitochondria.
Thus, a flavoprotein-containing oxidase whose activity is controlled by
cytosolic NAD(H) redox and PO2 appears to be an
important source of O2- production
in bovine cardiac myocytes.
Bovine calf cardiac myocytes have previously been shown to express a
basal intracellular source of production of
O2- that is detected by lucigenin-elicited
CL.7 This basal production of
O2- is shown in the present study to be
modulated by changes in PO2, increased in the
presence of 10 mmol/L lactate, and inhibited slightly by 10
mmol/L pyruvate potentially through the hypothesized mechanisms
included in the model in Fig 6. Since the major tissue
metabolites of lactate are pyruvate and NADH, lactate
deydrogenase-derived NADH is likely to be the key metabolite of lactate
that contributes to the generation of O2-.
Consistent with this role for cytosolic NADH in the
production of O2- is the
observation in this study in the homogenate of cardiac
myocytes that NADH oxidase is a major source of
O2- production. Previous studies
have reported the detection of an
O2--producing NADH oxidase activity in
cardiac muscle, which has been claimed to have the unusual property
that it is a mitochondrial system that uses extramitochondrial
NADH.16 We suspect that this previously detected
O2--producing NADH oxidase activity is
similar to the NADH oxidase identified in the present study.
However, since O2- production by
both of these NADH oxidase systems is not altered by rotenone, it is
likely that these oxidases are not linked to the mitochondrial NADH
dehydrogenase, which is thought to utilize extramitochondrial NADH.
This interpretation is based on more recent studies from the same
group17 indicating that the NADH dehydrogenase activity
linked to extramitochondrial NADH is inhibited by rotenone. The
inhibitory effects of DPI on
O2- production in intact and
homogenized cardiac myocytes are consistent with a
flavoprotein component in the detected
O2--producing oxidase. Our previous studies
on the properties and subcellular localization of
O2- production by a lactate and
cytosolic NADH redox–regulated NADH oxidase in bovine
pulmonary arterial smooth muscle are
consistent with this activity originating from a flavoprotein
and b558-type cytochrome containing microsomal
electron transport chain.18 19 On the basis of the results
of the present study, cardiac myocytes appear to have a similar
microsomal NADH-dependent O2--producing
electron transport chain containing a cytochrome
b558 whose activity is regulated by cytosolic
NAD(H) redox.
|
In the present study, attempts were made to detect several other
potential sources of O2- production
in the homogenate of bovine cardiac myocytes. It is well
established that exposure of cardiac muscle to H/R or I/R can lead to
conversion of xanthine dehydrogenase to xanthine
oxidase.1 2 23 Since hypoxia and ischemia
also seem to generate the substrates hypoxanthine and xanthine that are
required for the expression of xanthine oxidase activity, xanthine
oxidase appears to become a major source of
O2- in the myocardium upon
reoxygenation or reperfusion.23 24
However, the results of our previous study in intact bovine cardiac
myocytes did not detect a hypoxanthine-elicited increase in
O2- production either before or
after H/R.7 Consistent with these previous
observations and the known specific localization of xanthine
dehydrogenase/oxidase to endothelium,25
the present study did not detect evidence for xanthine oxidase
activity in homogenized myocytes. Since NADPH oxidases and
arachidonic acid–metabolizing enzymes have also been
shown to be potential sources of O2-
production,26 27 28 experiments in the present
study also examined the importance of these systems as myocyte sources
of O2- generation. In the calf myocyte
homogenate, the presence of 100 µmol/L NADPH was
observed to elicit a small but statistically significant increase in
lucigenin-dependent CL. Arachidonic acid plus NADPH was
used to detect O2- generation by enzymes
such as cyclooxygenase and
lipoxygenase.28 Under these conditions,
arachidonic acid did not cause a further increase in
lucigenin CL. Although the origin of the minor NADPH oxidase activity
that was detected was not further examined, it is possible that this
activity originates from utilization by NADH oxidases of NADPH as a
substrate or from NADPH oxidases that remain to be identified.
Observations made in the present study are consistent with
an absence of evidence in the literature for xanthine oxidase,
arachidonic acid–metabolizing enzymes, and NADPH
oxidases as major sources of production of
O2- in cardiac myocytes.
Mitochondria are considered to be an important source of generation of
O2- in cardiac myocytes.4 5 6 14
This concept is supported by observations in the present study on
the detection of H2O2 release from mitochondria
under conditions that maximize intramitochondrial generation of
O2- (succinate+antimycin15 ).
These conditions are thought to maximize
O2- production as a result of
blocking by antimycin of electron flow in the mitochondrial electron
transport chain in a manner that permits succinate dehydrogenase to
transfer electrons in a forward and reverse direction to the previously
identified key sites of O2-
production associated with NADH dehydrogenase (complex I) and
ubiquinone–cytochrome b.14 15 It should be
noted that our method of detection of mitochondrial
H2O2 release is expected to markedly
underestimate the actual rates of mitochondrial
O2- and H2O2
production, because it does account for O2
metabolites that are metabolized within the mitochondria. Previous
studies on mitochondrial NADH–dependent
O2- production indicate that
inhibitors that block electron transport at sites proximal
(rotenone) and distal (antimycin) to the location of electron transfer
to O2 cause decreased and increased production of
O2-, respectively.14 15 Thus,
O2- production by the
rotenone-inhibitable NADH dehydrogenase suggested to be an inner
mitochondrial membrane enzyme utilizing extramitochondrial (cytosolic)
NADH16 17 would be expected to be altered by the presence
of rotenone. In the present study, rotenone was found not to
significantly alter the level of lucigenin-detectable
O2- observed in the presence of lactate or
NADH in intact or homogenized myocytes, respectively. The
observed minor stimulating effects of rotenone and antimycin on basal
myocyte CL and the apparent suppression of the increase caused by
lactate in the presence of these inhibitors may originate
from potential effects of these electron transport
inhibitors on the control of cytosolic NAD(H) redox by
mitochondrial function, as has been observed in our previous studies on
calf pulmonary arterial smooth
muscle.12 We have previously suggested that lucigenin
appears to be a poor detector of intramitochondrial
O2-.8 Data in the present
study indicate that lucigenin CL was not increased by the
intramitochondrial source of O2- that
results in the mitochondrial release of H2O2
that is detectable as catalase-inhibitable increases in luminol CL.
Thus, the major source of lucigenin-detectable
O2- appears to originate from an NADH
oxidase activity that is not linked to intramitochondrial
O2- production.
The lucigenin-detectable source of production of
O2- observed in intact cardiac myocytes is
suppressed by hypoxia, and posthypoxic
reoxygenation elicits a transient
overproduction of O2-. A key role
for NADH oxidase in these PO2-elicited changes
in O2- detection is supported by the
inhibition by DPI and the enhancement by lactate of the measured levels
of lucigenin CL under these conditions. Although 10 mmol/L
pyruvate was observed to cause a small decrease in lucigenin CL under
basal conditions, it did not significantly alter the transient
overproduction of O2- observed
during posthypoxic reoxygenation. Our studies on the
PO2 dependence of
O2- production at constant levels
of NADH by a microsomal preparation of NADH oxidase isolated from
bovine pulmonary arterial smooth muscle determined
that it is suppressed by hypoxia and that posthypoxic
reoxygenation elicits a transient
overproduction of
O2-.18 Thus,
O2- production by the microsomal
NADH oxidase is suppressed by decreases in
physiological levels of
PO2, and a transient overproduction of
O2- during posthypoxic
reoxygenation is a property of the effects of
hypoxia on this electron transport chain. Isolated bovine
pulmonary19 and coronary9
arterial smooth muscle show
PO2-elicited changes in lucigenin-detectable
O2- that appear to originate from NADH
oxidase upon exposure to H/R and are similar to the responses observed
in cardiac myocytes and pulmonary arterial
microsomes containing NADH oxidase activity. Interestingly, a
comparison with O2- production by
previously studied bovine vascular smooth muscle
preparations8 9 19 suggests that bovine cardiac myocytes
have an 10-fold greater level of NADH oxidase activity and a
markedly increased spectral absorbance of microsomal cytochrome
b558 and that the myocytes generally show a
greater basal level of lucigenin CL, particularly after exposure to
H/R. On the basis of studies in a variety of tissue preparations,
including cardiac muscle, H/R and I/R also appear to cause the
accumulation of tissue lactate and an increase in the levels of
cytosolic NADH.29 30 Thus, increased levels of lactate and
cytosol-ic NADH that accumulate during the hypoxic exposure are
also likely to contribute to the transient overproduction of
O2- observed in bovine cardiac myocytes
during posthypoxic reoxygenation.
Experiments in the present study have identified an NADH oxidase
whose activity appears to be controlled by cytosolic NADH as an
important novel source of O2-
production in bovine cardiac myocytes. As hypothesized by
interactions shown in the model in Fig 6, the activity of this system
seems to be controlled in myocytes by changes in
PO2 and lactate in a manner suggesting that
O2- production by this NADH oxidase
may be of importance in signaling processes involving reactive
O2 species in cardiac myocytes. NADH oxidase–derived
O2- production may also have an
important role in the injury caused by exposure of cardiac muscle to
I/R, because the accumulation of tissue lactate and the associated
increase in cytosolic NADH have previously been suggested to actively
participate in metabolic processes that contribute to
tissue injury under these conditions.29 30 Heart failure
is an additional pathophysiological state in which
NADH oxidase may participate in increased myocardial
O2- production, because we have
observed elevated levels of lucigenin CL and NADH oxidase activity in
myocytes isolated from failing human and canine
hearts.31 32
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by grants HL-31069 and HL-43023 from the National Heart, Lung, and Blood Institute.
|
Footnotes |
|---|
Presented in part at the Experimental Biology '95 Conference, Atlanta, Ga, April 9-13, 1995, and published in abstract form (FASEB J. 1995;9:A12).
Received August 6, 1996; revision received December 16, 1996; accepted January 20, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol. 1988;255:H1269-H1275.
2. Hearse DG, Manning AS, Downey JM, Yellon DM. Xanthine oxidase: a critical mediator of myocardial injury during ischemia and reperfusion. Acta Physiol Scand Suppl. 1986;548:65-78.[Medline] [Order article via Infotrieve]
3.
Bolli R. Mechanism of myocardial
`stunning.' Circulation. 1990;82:723-738.
4.
Ambrosio G, Zweier JL, Duilio C, Kuppusamy P, Santoro
G, Elia PP, Tritto I, Cirillo P, Condorelli M, Chiariello M, Flaherty
JT. Evidence that mitochondrial respiration is a source of
potentially toxic oxygen free radicals in intact rabbit hearts
subjected to ischemia and reflow. J Biol
Chem. 1993;268:18532-18541.
5. Shlafer M, Myers CL, Adkins S. Mitochondrial hydrogen peroxide generation and activities of glutathione peroxidase and superoxide dismutase following global ischemia. J Mol Cell Cardiol. 1987;18:1195-1206.
6. Turrens JF, Beconi M, Barilla J, Chavez UB, McCord JM. Mitochondrial generation of oxygen radicals during reoxygenation of the ischemic tissues. Free Radic Res Commun. 1991;12-13:681-689.
7. Kaminski PM, Wolin MS. Hypoxia increases superoxide anion production from bovine coronary microvessels, but not cardiac myocytes, via increased xanthine oxidase. Microcirculation. 1994;1:231-236.[Medline] [Order article via Infotrieve]
8.
Mohazzab-H KM, Wolin MS. Sites of superoxide
anion production in calf pulmonary arterial
smooth muscle. Am J Physiol. 1994;267:L815-L822.
9.
Mohazzab-H KM, Fayngersh RP, Kaminski PM, Wolin
MS. Oxygen-elicited responses in calf coronary arteries:
role of H2O2 production via
NADH-derived superoxide. Am J Physiol. 1996;270:H1044-H1053.
10.
Mohazzab-H KM, Kaminski PM, Wolin MS.
NADH-oxidoreductase is a major source of superoxide anion in bovine
coronary endothelium. Am J
Physiol. 1994;266:H2568-H2572.
11.
Omar HA, Mohazzab-H KM, Mortelliti MP, Wolin MS.
O2 dependent modulation of calf pulmonary artery
tone by lactate: role of H2O2 and cGMP.
Am J Physiol. 1993;264:L141-L145.
12. Wolin MS, Burke-Wolin TM, Kaminski PM, Mohazzab-H KM. Reactive oxygen species and vascular oxygen sensors. In: Weir EK, Archer SL, Reeves JT, eds. Nitric Oxide and Radicals in the Pulmonary Vasculature. Armonk, NY: Futura Publishing Co; 1996:245-263.
13. Wolin MS, Mortelliti MP, Omar HA. Lactate appears to produce a cGMP-mediated relaxation of isolated bovine coronary arteries via the intracellular generation of reactive O2 species. Circulation. 1990;82(suppl III):III-422. Abstract.
14. Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980;191:421-427.[Medline] [Order article via Infotrieve]
15. Forman HJ, Boveris A. Superoxide radical and hydrogen peroxide in mitochondria. In: Pryor WA, ed. Free Radicals in Biology, Vol 5. New York, NY: Academic Press; 1982:65-90.
16. Nohl H. A novel superoxide radical generator in heart mitochondria. FEBS Lett. 1987;214:269-273.[Medline] [Order article via Infotrieve]
17. Schonheit K, Nohl H. Oxidation of cytosolic NADH via complex I of heart mitochondria. Arch Biochem Biophys. 1996;327:319-323.[Medline] [Order article via Infotrieve]
18.
Mohazzab-H KM, Wolin MS. Properties of a
superoxide anion generating microsomal NADH-oxidoreductase, a potential
pulmonary artery PO2 sensor. Am
J Physiol. 1994;267:L823-L831.
19.
Mohazzab-H KM, Fayngersh RP, Kaminski PM, Wolin
MS. Potential role of NADH oxidoreductase-derived reactive
O2 species in the calf pulmonary
arterial PO2-elicited responses.
Am J Physiol. 1995;269:L637-L644.
20. Mohazzab-H KM, Wolin MS. Sites of superoxide anion production in calf pulmonary arterial smooth muscle. Am J Physiol. 1994;267:L815-L822.
21.
Paky A, Michael JR, Burke-Wolin T, Wolin MS, Gurtner
GH. Endogenous production of superoxide by
rabbit lungs: effects of hypoxia or metabolic
inhibitors. J Appl Physiol. 1993;74:2868-2874.
22. Thorpe GHG, Kricka L. Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods Enzymol. 1986;133:331-353.[Medline] [Order article via Infotrieve]
23.
Yia Y, Zweier JL. Substrate control of free
radical generation from xanthine oxidase in the
postischemic heart. J Biol Chem. 1995;270:18797-18803.
24. Saugstad OS. Hypoxanthine as an indicator of hypoxia: its role in health and disease through free radical production. Pediatr Res. 1988;23:143-150.[Medline] [Order article via Infotrieve]
25. Jarasch E-D, Bruder G, Heid HW. Significance of xanthine oxidase in capillary endothelial cells. Acta Physiol Scand Suppl. 1986;548:39-46.
26. Cross AR, Jones OTG. Enzymatic mechanisms of superoxide production. Biochim Biophys Acta. 1991;1057:291-298.
27.
Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM.
Generation of superoxide by purified brain nitric oxide
synthase. J Biol Chem. 1992;267:24173-24176.
28.
Kukreja RC, Kontos HA, Hess ML, Ellis EF. PGH
synthetase and lipoxygenase generate superoxide in the
presence of NADH or NADPH. Circ Res. 1986;59:612-619.
29.
Rovetto MJ, Lamberton WF, Neely JR. Mechanisms
of glycolytic inhibition in isolated rat hearts. Circ
Res. 1975;37:742-751.
30.
Neely JR, Crotyohann LW. Role of glycolytic
products in damage to ischemic myocardium:
dissociation of adenosine triphosphate levels and recovery of
function of reperfused ischemic hearts. Circ
Res. 1984;55:816-824.
31. Mohazzab-H KM, Zhang X, Kichuk MR, Michler R, Kaley G, Wolin MS. Potential sites of changes of superoxide production in failing and non-failing human cardiac myocytes. Circulation. 1995;92(suppl I):I-32. Abstract.
32. Mohazzab-H KM, Zhang X, Xu X, Hintze TH, Wolin MS. Evidence for an increase in superoxide anion generation in pacing-induced failing dog heart myocytes. FASEB J. 1996;10:A581. Abstract.
This article has been cited by other articles:
 |
|
 |
|
  T. Hayashi, C. Yamashita, C. Matsumoto, C.-J. Kwak, K. Fujii, T. Hirata, M. Miyamura, T. Mori, A. Ukimura, Y. Okada, et al. Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2197 - H2203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Cooper, A. Whaley-Connell, J. Habibi, Y. Wei, G. Lastra, C. Manrique, S. Stas, and J. R. Sowers Renin-angiotensin-aldosterone system and oxidative stress in cardiovascular insulin resistance Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2009 - H2023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Whaley-Connell, G. Govindarajan, J. Habibi, M. R. Hayden, S. A. Cooper, Y. Wei, L. Ma, M. Qazi, D. Link, P. R. Karuparthi, et al. Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E355 - E363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Zima and L. A. Blatter Redox regulation of cardiac calcium channels and transporters Cardiovasc Res, July 15, 2006; 71(2): 310 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, P. S. Gill, and W. J. Welch Oxygen availability limits renal NADPH-dependent superoxide production Am J Physiol Renal Physiol, October 1, 2005; 289(4): F749 - F753. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Wolin and S. A. Gupte Novel Roles for Nox Oxidases in Cardiac Arrhythmia and Oxidized Glutathione Export in Endothelial Function Circ. Res., September 30, 2005; 97(7): 612 - 614. [Full Text] [PDF]
|
|
|
|
|
|
S. Kinugawa, J. Zhang, E. Messina, E. Walsh, H. Huang, P. M. Kaminski, M. S. Wolin, and T. H. Hintze gp91phox-containing NAD(P)H oxidase mediates attenuation of nitric oxide-dependent control of myocardial oxygen consumption by ANG II Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H862 - H867. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Mallet, J. Sun, E. M. Knott, A. B. Sharma, and A. H. Olivencia-Yurvati Metabolic Cardioprotection by Pyruvate: Recent Progress Experimental Biology and Medicine, July 1, 2005; 230(7): 435 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Peng, X. Lu, and Q. Feng Pivotal Role of gp91phox-Containing NADH Oxidase in Lipopolysaccharide-Induced Tumor Necrosis Factor-{alpha} Expression and Myocardial Depression Circulation, April 5, 2005; 111(13): 1637 - 1644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kinugawa, Z. Wang, P. M. Kaminski, M. S. Wolin, J. G. Edwards, G. Kaley, and T. H. Hintze Limited Exercise Capacity in Heterozygous Manganese Superoxide Dismutase Gene-Knockout Mice: Roles of Superoxide Anion and Nitric Oxide Circulation, March 29, 2005; 111(12): 1480 - 1486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Labarthe, M. Khairallah, B. Bouchard, W. C. Stanley, and C. Des Rosiers Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1425 - H1436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Browe and C. M. Baumgarten Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl- Current Elicited by {beta}1 Integrin Stretch in Rabbit Ventricular Myocytes J. Gen. Physiol., August 30, 2004; 124(3): 273 - 287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Zima, J. A. Copello, and L. A. Blatter Effects of cytosolic NADH/NAD+ levels on sarcoplasmic reticulum Ca2+ release in permeabilized rat ventricular myocytes J. Physiol., March 15, 2004; 555(3): 727 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Meissner NADH, a New Player in the Cardiac Ryanodine Receptor? Circ. Res., March 5, 2004; 94(4): 418 - 419. [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Lee, I.-J. Kang, R. Bunger, and Y.-H. Kang Enhanced survival effect of pyruvate correlates MAPK and NF-{kappa}B activation in hydrogen peroxide-treated human endothelial cells J Appl Physiol, February 1, 2004; 96(2): 793 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kinugawa, H. Post, P. M. Kaminski, X. Zhang, X. Xu, H. Huang, F. A. Recchia, M. Ochoa, M. S. Wolin, G. Kaley, et al. Coronary Microvascular Endothelial Stunning After Acute Pressure Overload in the Conscious Dog Is Caused by Oxidant Processes: The Role of Angiotensin II Type 1 Receptor and NAD(P)H Oxidase Circulation, December 9, 2003; 108(23): 2934 - 2940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Moritz, C. Monteil, M. Isabelle, F. Bauer, S. Renet, P. Mulder, V. Richard, and C. Thuillez Role of reactive oxygen species in cocaine-induced cardiac dysfunction Cardiovasc Res, October 1, 2003; 59(4): 834 - 843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Pacher, L. Liaudet, P. Bai, J. G. Mabley, P. M. Kaminski, L. Virag, A. Deb, E. Szabo, Z. Ungvari, M. S. Wolin, et al. Potent Metalloporphyrin Peroxynitrite Decomposition Catalyst Protects Against the Development of Doxorubicin-Induced Cardiac Dysfunction Circulation, February 18, 2003; 107(6): 896 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Li, N. P. Gall, D. J. Grieve, M. Chen, and A. M. Shah Activation of NADPH Oxidase During Progression of Cardiac Hypertrophy to Failure Hypertension, October 1, 2002; 40(4): 477 - 484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. H. Khadour, D. Panas, P. Ferdinandy, C. Schulze, T. Csont, M. M. Lalu, S. M. Wildhirt, and R. Schulz Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1108 - H1115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Mizutani, S. Tada-Oikawa, Y. Hiraku, S. Oikawa, M. Kojima, and S. Kawanishi Mechanism of Apoptosis Induced by a New Topoisomerase Inhibitor through the Generation of Hydrogen Peroxide J. Biol. Chem., August 16, 2002; 277(34): 30684 - 30689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ahmad, Y. Zhang, Y. Zhang, C. Papharalambus, and R. W. Alexander Role of Isoprenylcysteine Carboxyl Methyltransferase in Tumor Necrosis Factor-{alpha} Stimulation of Expression of Vascular Cell Adhesion Molecule-1 in Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 759 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ozcan, M. Bienengraeber, P. P. Dzeja, and A. Terzic Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H531 - H539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Bendall, A. C. Cave, C. Heymes, N. Gall, and A. M. Shah Pivotal Role of a gp91phox-Containing NADPH Oxidase in Angiotensin II-Induced Cardiac Hypertrophy in Mice Circulation, January 22, 2002; 105(3): 293 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. MacCarthy, D. J. Grieve, J.-M. Li, C. Dunster, F. J. Kelly, and A. M. Shah Impaired Endothelial Regulation of Ventricular Relaxation in Cardiac Hypertrophy: Role of Reactive Oxygen Species and NADPH Oxidase Circulation, December 11, 2001; 104(24): 2967 - 2974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. D. Mongan, J. Capacchione, J. L. Fontana, S. West, and R. Bunger Pyruvate improves cerebral metabolism during hemorrhagic shock Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H854 - H864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Bassenge, O. Sommer, M. Schwemmer, and R. Bunger Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2431 - H2438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ferdinandy, H. Danial, I. Ambrus, R. A. Rothery, and R. Schulz Peroxynitrite Is a Major Contributor to Cytokine-Induced Myocardial Contractile Failure Circ. Res., August 4, 2000; 87(3): 241 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Griendling, D. Sorescu, and M. Ushio-Fukai NAD(P)H Oxidase : Role in Cardiovascular Biology and Disease Circ. Res., March 17, 2000; 86(5): 494 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. Becker, T. L. vanden Hoek, Z.-H. Shao, C.-Q. Li, and P. T. Schumacker Generation of superoxide in cardiomyocytes during ischemia before reperfusion Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2240 - H2246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Xie, P. Kometiani, J. Liu, J. Li, J. I. Shapiro, and A. Askari Intracellular Reactive Oxygen Species Mediate the Linkage of Na+/K+-ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes J. Biol. Chem., July 2, 1999; 274(27): 19323 - 19328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Vanden Hoek, L. B. Becker, Z. Shao, C. Li, and P. T. Schumacker Reactive Oxygen Species Released from Mitochondria during Brief Hypoxia Induce Preconditioning in Cardiomyocytes J. Biol. Chem., July 17, 1998; 273(29): 18092 - 18098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Duranteau, N. S. Chandel, A. Kulisz, Z. Shao, and P. T. Schumacker Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes J. Biol. Chem., May 8, 1998; 273(19): 11619 - 11624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-Y. Sohn, M. Keller, T. Gloe, H. Morawietz, U. Rueckschloss, and U. Pohl The Small G-protein Rac Mediates Depolarization-induced Superoxide Formation in Human Endothelial Cells J. Biol. Chem., June 16, 2000; 275(25): 18745 - 18750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Miramar, P. Costantini, L. Ravagnan, L. M. Saraiva, D. Haouzi, G. Brothers, J. M. Penninger, M. L. Peleato, G. Kroemer, and S. A. Susin NADH Oxidase Activity of Mitochondrial Apoptosis-inducing Factor J. Biol. Chem., May 4, 2001; 276(19): 16391 - 16398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Li, F.-X. Yi, J. L. Spurrier, C. A. Bobrowitz, and A.-P. Zou Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney Am J Physiol Renal Physiol, June 1, 2002; 282(6): F1111 - F1119. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||