Pulmonary reduction of an intravascular redox polymer
Said H.
Audi1,2,
Robert
D.
Bongard3,
Yoshiyuki
Okamoto4,
Marilyn P.
Merker1,5,6,
David L.
Roerig5,6,7, and
Christopher A.
Dawson1,2,3,5,6,7
1 Department of Biomedical Engineering, Marquette
University, Milwaukee 53201-1881; Departments of 2 Pulmonary
and Critical Care Medicine, 3 Physiology,
5 Anesthesiology, and 6 Pharmacology/Toxicology, Medical
College of Wisconsin, Milwaukee 53226; 7 Zablocki Veterans
Affairs Medical Center, Department of Veterans Affairs, Milwaukee,
Wisconsin 53295; and 4 Department of Chemistry, Polytechnic
University, Brooklyn, New York 11201
 |
ABSTRACT |
Pulmonary
endothelial cells in culture reduce external electron acceptors via
transplasma membrane electron transport (TPMET). In studying
endothelial TPMET in intact lungs, it is difficult to exclude
intracellular reduction and reducing agents released by the lung.
Therefore, we evaluated the role of endothelial TPMET in the reduction
of a cell-impermeant redox polymer, toluidine blue O polyacrylamide
(TBOP+), in intact rat lungs. When added to the perfusate
recirculating through the lungs, the venous effluent TBOP+
concentration decreased to an equilibrium level reflecting
TBOP+ reduction and autooxidation of its reduced (TBOPH)
form. Adding superoxide dismutase (SOD) to the perfusate increased the
equilibrium TBOP+ concentration. Kinetic analysis indicated
that the SOD effect could be attributed to elimination of the
superoxide product of TBOPH autooxidation rather than of superoxide
released by the lungs, and experiments with lung-conditioned perfusate
excluded release of other TBOP+ reductants in sufficient
quantities to cause significant TBOP+ reduction. Thus the
results indicate that TBOP+ reduction is via TPMET and
support the utility of TBOP+ and the kinetic model for
investigating TPMET mechanisms and their adaptations to physiological
and pathophysiological stresses in the intact lung.
lung metabolism; oxidation-reduction; mathematical modeling; superoxide; ascorbate
 |
INTRODUCTION |
TRANSPLASMA MEMBRANE
ELECTRON TRANSPORT (TPMET) systems exist in various cell types
(12, 18, 19, 21, 30, 31, 33, 39, 41) and have been
implicated in a wide range of functions (5, 6, 11, 12, 18, 20,
21, 23, 28, 29, 32, 33, 35, 37, 39). Vascular endothelial TPMET
(7-9, 15, 26, 27) is unique in its potential
for influencing plasma redox status. For example, vascular endothelial
cells, along with monocytes, are the key cell types involved in
atherosclerotic plaque formation (38, 39). They are
commonly thought to promote plasma lipoprotein oxidation
(39), but in the absence of free transition metal ions,
they have an antioxidant influence on plasma lipoproteins (18,
39). Both the prooxidant and antioxidant effects can be
explained by TPMET systems that can either promote oxidation by
reducing metal ions (15, 18, 40) or protect lipoproteins
from oxidation by regenerating lipoprotein antioxidants (2, 8,
14, 19, 30). The pulmonary endothelium is particularly interesting in this regard because it is a large reactor surface upstream from the vulnerable systemic arterial system wherein the toxic
effects of oxidized lipoproteins are manifest (38), but
the local endothelial surface area-to-blood flow ratio is relatively
very small. Additional consequences of pulmonary endothelial TPMET may
include the pulmonary endothelial targeting of redox-active toxins
(7, 19).
Thiazine electron acceptors have been used as probes for studying
pulmonary endothelial TPMET because their redox status is easily
measured (4, 7, 14, 26) and because they are electron
acceptors (i.e., substrate analogs) for reductases acting on many
physiologically, pharmacologically, and toxicologically important
redox-active compounds (16). The TPMET system demonstrated in pulmonary arterial endothelial cells grown in culture undoubtedly contributes to the reduction of thiazine electron acceptors on passage
through the lungs (4, 7, 14, 26). However, the role of
endothelial TPMET within intact lungs in the overall reduction process
is more difficult to assess than in cell culture. This is, in part,
because the cells are not directly accessible, which contributes to the
difficulty in demonstrating whether a given electron acceptor probe
might have been taken up and reduced intracellularly during transit
through the lungs (4, 7) and/or whether some short-lived
reducing agent might have been released by the lungs (3, 10, 22,
32). Therefore, the objective of the present study was to
evaluate the role of endothelial TPMET in the reduction of thiazine
electron acceptors within the lungs, and, in doing so, to develop a
methodological basis for future investigations into the physiological
and pathophysiological adaptations of pulmonary endothelial TPMET.
Experiments were carried out with isolated rat lungs perfused with an
electron acceptor, toluidine blue O (TBO) covalently bound to a
polyacrylamide (TBOP) polymer (9), that was too large to
enter the cells or pass through the capillary wall in a significant
quantity over the time course of the experiment. Thus reduction of the
TBOP polymer from its blue oxidized form (TBOP+) to its
colorless reduced form (TBOPH) on passage through the lungs is
indicative of reduction within the vessel lumen. The possibility that
some short-lived and/or low molecular weight reductant(s) released by
the lungs might contribute to TBOP+ reduction was also
evaluated, and a kinetic analysis of the data was carried out to assist
in their interpretation.
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EXPERIMENTAL METHODS |
The isolated rat lung preparation used has been previously
described (25). Each rat [314 ± 42 (SD) g] was
anesthetized (pentobarbital sodium 0.040 mg/g body wt ip). The trachea
was clamped, and the chest was opened. Heparin (200 IU in 0.2 ml) was
injected into the right ventricle. The pulmonary artery, left atrium,
and trachea were cannulated with polyethylene tubing (1.67-mm ID,
2.42-mm OD). The lungs were removed from the chest, and the respective cannulas were attached to the ventilation and perfusion system. The
physiological salt solution (PSS) perfusate was maintained at 35°C
and contained (in mM) 4.7 KCl, 2.51 CaCl2, 1.19 MgSO4, 2.5 KH2PO4, 118 NaCl, 25 NaHCO3, 5.5 glucose, and 5% bovine serum albumin. The
initial volume (~40 ml) of perfusate pumped (MasterFlex roller pump)
through the lungs was discarded until the lungs and venous effluent
were visually clear of blood. The venous effluent was then sent into a
reservoir, from which it was pumped back into the pulmonary artery at a
flow rate of 10 ml/min. The total volume of the recirculating perfusion
system including the lung vascular volume [~0.85 ml
(14)] was ~16 ml. The lungs were ventilated with 8 mmHg
end-inspiratory and 3 mmHg end-expiratory pressures at 40 breaths/min
and a gas composition of 15% O2 and 6% CO2 in N2, giving perfusate PO2,
PCO2, and pH values of 139 ± 11 (SD) Torr, 35 ± 3 Torr, and 7.36 ± 0.02, respectively. The left
atrial pressure was set at atmospheric pressure. The pulmonary arterial pressure relative to the left atrium was continuously monitored during
the course of the experiments and averaged 6.5 ± 0.8 (SD) mmHg at
the beginning and 5.6 ± 0.9 (SD) mmHg at the end of the perfusion
periods, delimiting the experimental protocols described below. At the
end of the experiments, the lungs were weighed, dried, and reweighed.
The lung wet weight averaged 1.45 ± 0.31 (SD) g, with a
wet-to-dry weight ratio of 5.57 ± 0.76 (SD).
The TBOP polymer was synthesized as previously described
(9). For the batch used in these experiments, the average
molecular weight was ~35,000, and molecular weight distribution was
such that the fractions passing through 3,500, 10,000 and 30,000 molecular weight cutoff filters were not detectable, 3.5%, and 31%,
respectively. There were ~32 nmol redox-active TBO moieties/mg,
determined as previously described (9). Absorption spectra
of the oxidized form (TBOP+) and the fully reduced form
(TBOPH), produced by anaerobic reduction with xanthine oxidase plus
hypoxanthine in a Thunberg cuvette, are shown in Fig.
1.

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Fig. 1.
Absorption spectra for the oxidized (TBOP+)
and reduced (TBOPH) forms of toluidine blue O polyacrylamide (TBOP).
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To study TBOP disposition in the perfused lungs, the recirculating
perfusate volume was decreased to 8 ml and then immediately replenished
at time 0 by adding 8 ml of perfusate containing 4 mg/ml of
TBOP+ equilibrated with the respiratory gas mixture at the
perfusion system temperature. Thus the initial concentration in the
total perfusate volume was 2 mg/ml. To evaluate the contribution of superoxide and ascorbate released by the lungs to the reduction process, in some experiments, the 8 ml containing the TBOP+
also contained either superoxide dismutase (SOD; 2,080 U) or ascorbate
oxidase (AO; 144 U). The TBOP-containing perfusate was recirculated
through the lungs for 20-60 min. At the specific times indicated
in EXPERIMENTAL RESULTS, 1 ml of lung effluent was
collected for spectrophotometric analysis (Beckman DU 7400). To
determine the TBOP+ concentration in a sample, it was
immediately centrifuged (5,500 g for 40 s in a Costar
microcentrifuge), and absorbance was measured at 590 and 750 nm. The
centrifugation was carried out to eliminate turbidity due to any red
cells that might not have been completely removed during the washing
period, and the absorbance at 750 nm was subtracted from that at 590 nm
as an additional check on turbidity. The sample was then returned to
the recirculating perfusion system. To determine the amount of TBOPH
and the rate of TBOPH autooxidation in selected samples, the samples
were placed immediately in the spectrophotometer and absorbance was
recorded continuously until a steady level was achieved by a maximum of
~40 min. At that point, the sample was centrifuged, and the
absorbance was measured again.
Any possible contribution of the perfusate and perfusion system tubing
to the observed TBOP+ reduction under the experimental
conditions was evaluated by carrying out the same experiments except
with the rat lungs replaced by an additional length of tubing (sham
lung) of approximately equal volume.
Additional experiments were carried out with the lungs in which the 8 ml added at time 0 was plain PSS containing no TBOP. These
TBOP-free experiments were used to obtain conditioned PSS to evaluate
the possibility that recirculation through the lungs might result in
spectral properties interfering with the TBOP+ measurement
and/or the possibility that the lungs might release some
TBOP+ reductant(s) into the perfusate. Samples obtained at
the usual sampling times had no above-blank absorbance at 590 nm,
ruling out the first of these possibilities. The possible release of a
TBOP+ reducing agent was examined by adding
TBOP+ (2 mg/ml; note that this and the subsequently
indicated reagent concentrations are the initial concentrations in
total reaction mixture volume) to the samples removed after 0, 10, and
30 min of recirculation through the lungs, and the absorbance was
continuously measured for the subsequent 25 min or longer, if
necessary, to reach steady level. Because of the known release of
ascorbate by perfused lungs (1, 10), the same procedure
was also carried out with AO (9 U/ml) added to the conditioned medium,
and the conditioned perfusate samples were also assayed for ascorbate as previously described (10).
For each of the above experiments, a sample of the PSS containing 4 mg/ml of TBOP+ that was added to the lung perfusion circuit
was diluted 1:1 with plain PSS. The absorbances of the fully oxidized
(TBOP+) and fully reduced (TBOPH) mixtures served to set
the range for calculating the concentration of TBOP+ in the
lung and sham effluents and conditioned perfusate samples and the
fraction of TBOP+ recovered in the perfusate at the end of
the recirculation period after complete reoxidation of any TBOPH formed
during the lung perfusion. The calculated recovery of TBOP+
for the lung experiments (n = 15) was 102.2 ± 2.9% (SD).
To evaluate the effect of the concentration of SOD used in the lung
experiments on the superoxide dismutation rate, the
superoxide-generating system xanthine oxidase (0.04 U/ml) plus
hypoxanthine (70 µM), the superoxide detector ferricytochrome
c (23.5 µM), and catalase (7,950 U/ml) were added to PSS
with and without SOD (130 U/ml) present. The resulting evolution of
ferrocytochrome c was measured spectrophotometrically (550 nm) (24).
The possible role of hydrogen peroxide (H2O2)
as a TBOPH oxidant was evaluated by adding ascorbate (30 µM) and
TBOP+ (2 mg/ml) to the PSS solution while monitoring
TBOP+ absorbance. Two minutes after the ascorbate was
added, H2O2 (4 mM) was added followed by
horseradish peroxidase (4.6 U/ml) 3 min later.
Because nitric oxide (NO) is another redox-active substance released by
the lung endothelium, the possibility that NO might be a
TBOP+ reductant was evaluated by adding TBOP+
(2 mg/ml) to a buffer solution that was equilibrated with 200 parts/million NO in N2 in a Thunberg cuvette. The sample
absorbance was measured for 5 min, and no TBOP+ reduction
could be detected.
 |
EXPERIMENTAL RESULTS |
Figure 2, left, shows
lung and sham effluent TBOP+ fractions after the addition
of TBOP+, TBOP+ plus SOD, or TBOP+
plus AO to the recirculating PSS perfusate. The 100% level is the
initial amount of TBOP+ added to the recirculating
perfusate at time 0 divided by the perfusate volume and is
approximately equal to the TBOP+ concentration entering the
lung or lung replacement tubing (sham) at time 0. Initially,
the effluent had no TBOP+ until the first lung or sham
transit after time 0. Then the sham effluent
TBOP+ fraction jumped to 100% and remained virtually
constant for the duration of the recirculation period. On the other
hand, the lung effluent TBOP+ fraction jumped to a level
somewhere below 100% as a result of reduction in the lungs. This was
followed by a decrease in the lung effluent TBOP+ fraction
to a plateau level, which was achieved by 15 min under all the
experimental conditions studied. The plateau level appears to be
determined by two competing processes, namely TBOP+
reduction in the lungs and TBOPH autooxidation throughout the perfusion
system. This is revealed by the data in Fig. 2, right, which
show the TBOP+ fraction versus time in the lung and sham
effluent samples after the samples had been removed from the perfusion
system. When the TBOPH formed during circulation through the lungs was
removed from the lung perfusion circuit, it fully autooxidized within 30 min as indicated by the increase in the TBOP+ fraction
to nearly 100%. Thus the TBOP+ fraction in Fig. 2,
left, is that resulting from both TBOP+
reduction on passage through lungs and the simultaneous autooxidation of TBOPH, whereas the TBOP+ fraction in Fig. 2,
right, reflects only the TBOPH autooxidation independent of
lung TBOP+ reduction. Within an experiment, TBOPH
autooxidation rates in lung effluent samples removed from the perfusion
system at times ranging between 10 and 60 min during the recirculation
period were not significantly different. For each lung, the
autooxidation data obtained at the different sample times were averaged
to provide a mean autooxidation data set for that lung. Figure 2,
right, is the average of these autooxidation data sets from
all lungs for a given experimental condition. In the presence of SOD,
the lung effluent plateau TBOP+ fraction was higher and
autooxidation of TBOPH was more rapid than in the absence of SOD. AO,
on the other hand, had little effect. The sham effluent
TBOP+ fraction was virtually 100% during perfusion and
after removal from the perfusion system under all experimental
conditions studied. Only the PSS sham TBOP+ data are shown
in Fig. 2 because data with and without either SOD or AO added to the
PSS perfusate were virtually superimposed. Quantitative descriptors of
the data in Fig. 2 are given in Table 1.

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Fig. 2.
Left: lung and sham effluent TBOP+
concentrations expressed as a percentage of the initial concentration
of TBOP+ ([TBOP+]) added to the perfusate vs.
recirculation time with no additions other than TBOP [physiological
salt solution (PSS)] and with superoxide dismutase (SOD) or ascorbate
oxidase (AO) added. Right: [TBOP+] vs. time in
effluent samples after they had been removed from the perfusion system.
Only the PSS sham TBOP+ data are shown because PSS,
PSS+SOD, and PSS+AO data are virtually superimposed. Values are
means ± SE.
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To determine whether some relatively stable TBOP+
reductant(s) might be released from the lungs into the perfusate, the
lungs were perfused in the same manner as in Fig. 2 but with no
TBOP+ added to the perfusate so that the lung-conditioned
perfusate could be sampled. Figure 3
shows that when TBOP+ was added to the conditioned
perfusate removed from the recirculating system after 10 or 30 min of
recirculation, the TBOP+ concentration fell and then
returned to its initial level, indicating that a TBOP+
reducing agent did, in fact, accumulate in the perfusate. The reduction
was not observed in the parallel samples to which AO had been added.
Thus ascorbate accumulation in the conditioned perfusate appears to
quantitatively account for the TBOP+ reduction by the
conditioned perfusate.

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Fig. 3.
[TBOP+] vs. time after TBOP+
addition to conditioned perfusate samples with (+; right) or
without AO ( ; left) present. Model represents Eqs.
9-11 fit to the data.
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The above results raise questions as to the implications of the
substantial SOD effect with regard to a possible role for superoxide
released from the lungs as a TBOP+ reductant and the
apparent discrepancy between the effects of AO on TBOP+
reduction in the lungs and conditioned medium. We addressed these questions with the aid of a kinetic model as described in KINETIC MODEL.
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KINETIC MODEL |
The model derivation begins with the stoichiometric expressions,
included to account for the effects of the various experimental conditions on the disposition of TBOP within the lungs and perfusate. In the model, the lungs can contribute to TBOP+ reduction
via three mechanisms addressed in the experiments as depicted in Fig.
4. The lungs can directly reduce
TBOP+ via TPMET
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(a)
|
where E is thiazine reductase; D+ and DH are the
oxidized and reduced forms, respectively, of the intra-cellular
electron donor [e.g., NADH/NADPH (13, 34)]. The lungs
can also release superoxide and/or ascorbate into the perfusate at
constant rates (kso and
kas, respectively; in nmol/min), which, in turn,
reduce TBOP+ via
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(b)
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(c)
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where AH2 and A are ascorbate and
dehydroascorbate, respectively, and kx
represents all reaction rate constants.

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Fig. 4.
Schematic diagram depicting mechanisms of
TBOP+ reduction to TBOPH within the capillary region of the
kinetic model. AH2 and A, ascorbate and dehydroascorbate,
respectively; DH and D+, reduced and oxidized forms of the
intracellular electron donor, respectively; TPMET, endothelial
transplasma membrane electron transport system represented by thiazine
reductase (E) in reaction a.
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Reactions b and c and superoxide dismutation via
reaction d occur within the perfusate independently of the
lungs per se
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(d)
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The kinetic model assumes that the H2O2
product of reaction d does not react with TBOP+
or TBOPH under the experimental conditions studied. This assumption is
supported by the data in Fig. 5, which
show that the addition of H2O2 had little
effect on TBOPH oxidation rate. H2O2 is, in fact, an oxidant relative to TBOPH, but the reaction is quite slow in
the absence of peroxidase activity for which TBOPH is a rather good
substrate (Fig. 5).

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Fig. 5.
[TBOP+] vs. time with sequential additions
of ascorbate, hydrogen peroxide (H2O2), and
horseradish peroxidase at times indicated (arrows).
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Reactions b-d take place throughout
the perfusion system volume that has two components, a lung capillary
volume and a noncapillary volume made up of the reservoir, connecting
tubing, arteries, and veins. Reaction a occurs only within
the lung capillary volume.
The spatial and temporal variations in the concentrations of the
various species within the capillary and noncapillary volumes are
described by the following sets of partial (
) and ordinary (d)
differential equations based on the above reactions and the assumptions
that the concentrations of O2, DH, and H+
([O2], [DH], and [H+], respectively) were
constant under the study conditions and that within the pulmonary
capillary bed, the reduction rate of TBOP+ was too fast for
axial diffusion to dissipate axial concentration gradients of the
various chemical species, but the radial dimensions were so small that
no radial gradients develop.
Capillary volume.
The spatial and temporal variations of the concentrations of the
various species within the capillary volume are described by
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(1)
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(2)
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(3)
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(4)
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where [TBOPH]L(t,x),
[TBOP+]L(t,x),
[O
·]L(t,x),
and [AH2]L(t,x) are the
respective concentrations of TBOP+, TBOPH,
O
·, and AH2 at distance x from the capillary inlet and at time t; Qc and
W are the capillary volume and the average linear flow
velocity within Qc, respectively; kred = k1[E][DH], where [E] is the thiazine
reductase concentration; ko = k
2[O2]2;
k
o = k2[H+]; and
k
= k4[H+]2.
Noncapillary volume.
The temporal variations in the concentration of the various species in
the noncapillary volume are described by
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(5)
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(6)
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(7)
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(8)
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where [TBOPH]R(t),
[TBOP+]R(t),
[O
·]R(t), and
[AH2]R(t) are the respective
concentrations of TBOP+, TBOPH, O
·,
and AH2 in the noncapillary part of the perfusion system at
time t. QR and F are the combined volume of the
noncapillary part of the perfusion system and pump flow rate, respectively.
The model parameters are kred (ml/min), the
measure of TBOP+ reduction rate by thiazine reductase (to
put the units of kred into perspective,
kred is the lung perfusion rate that would
result in a 1/e fraction of TBOP+ being reduced
on a single passage through the lungs in the absence of any other
reactions); kso (nmol/min), the rate of
superoxide release by the lungs; ko
(min
1), the rate constant for TBOPH autooxidation;
k
o
(µM
2 · min
1), the rate constant
for superoxide-mediated TBOP+ reduction;
k
(µM
1 · min
1), the superoxide
dismutation rate constant; k3
(µM
1 · min
1), the rate constant
for TBOP+ reduction by ascorbate; and
kas (nmol/min), the rate for ascorbate release
by the lungs.
In what follows, parameter estimation was carried out with a
Levenberg-Marquardt optimization routine (4). At each
iteration, the relevant sets of equations were numerically solved using
the finite difference method (4) for the appropriate
initial and boundary conditions indicated in MODEL FIT AND
ESTIMATION OF MODEL PARAMETERS.
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MODEL FIT AND ESTIMATION OF MODEL PARAMETERS |
Because all of the model parameters are not separately
identifiable with the data from a single type of experiment, we used a
sequential approach for fitting the model described by Eqs. 1-8 to the data in Figs. 2 and 3. We began by
using the observation that when AO was added to the perfusate
recirculating through the lungs (PSS plus AO), the results were nearly
indistinguishable from the experiments with no enzyme added to the PSS
perfusate (Fig. 2, Table 1). Thus to simplify the model initially, the ascorbate contribution to TBOP+ reduction was set to zero.
The implications of this simplification are evaluated later.
In the presence of SOD, it was assumed that k
was fast enough that reactions b and d, which
account for TBOPH autooxidation and superoxide dismutation,
respectively, reduce to the unidirectional reaction
This assumption is supported by the data in Fig.
6 that evaluate the effect of SOD on the
superoxide dismutation rate with the ferricytochrome c-SOD
assay. The ratio of the initial rates of ferrocytochrome c
generation via the reduction of ferricytochrome c by the
hypoxanthine-xanthine oxidase system with and without the SOD present
(Fig. 6) provides a lower bound on the ratio of catalyzed to
uncatalyzed superoxide dismutation rates (Kd),
which was >2 × 102.

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Fig. 6.
Ferrocytochrome c concentration vs. time after
the addition of ferricytochrome c and hypoxanthine to PSS
with and without SOD present. Arrow, addition of xanthine oxidase.
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Under the above assumptions, the TBOP+ data obtained
with SOD present provide an estimate of the contribution of the
reductase (E) to TBOP+ reduction in the lungs. The
parameters identifiable from the data with SOD present were then
kred and ko, with the
other model parameters set to zero. Thus the model was fit
to the average TBOP+ data in Fig. 2, with SOD
present by solving Eqs. 1, 2, 5, and 6 with the initial conditions
[TBOPH]L(0,x) = [TBOP+]L(0,x) = 0, [TBOP+]R(0) = [TBOP+]0, and
[TBOPH]R(0) = 0 and the boundary
conditions [TBOPH]L(t,0) = [TBOPH]R(t) and
[TBOP+]L(t,0) = [TBOP+]R(t), where
[TBOP+]0 is the initial (time 0)
concentration of TBOP+. The estimated values were
kred = 1.73 ml/min and
ko = 0.316 min
1. The
estimated value of kred can be used to calculate
a TBOP+ reduction rate of 73 nmol/min from
kred[TBOP+]0.
With kred and ko known,
we proceeded to evaluate the contribution of superoxide released by the
lungs to TBOP+ reduction by fitting the model to the
average TBOP+ data in Fig. 2 obtained from the experiments
in which the lungs were perfused with no enzyme added. The identifiable
model parameters were then kso and
k
o, with the uncatalyzed superoxide
dismutation rate (k
) set to 12 µM
1 · min
1 as estimated by
Fridovich et al. (17). The model fit was obtained by
solving Eqs. 1-8 with the initial conditions
[TBOPH]L(0,x) = [O
·]L(0,x) = [TBOP+]L(0,x) = 0, [TBOP+]R(0) = [TBOP+]0, and
[TBOPH]R(0) = [O
·]R(0) = 0 and
the boundary conditions [TBOPH]L(t,0) = [TBOPH]R(t),
[O
·]L(0,x) = [O
·]R(t), and
[TBOP+]L(t,0) = [TBOP+]R(t). The estimated values
of the rate of superoxide release by the lungs
(kso) and the rate constant for
superoxide-mediated TBOP+ reduction
(k
o) were 26.2 nmol/min and 0.804 µM
2 · min
1, respectively. These
model fits are indicated in Fig. 7.

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Fig. 7.
Model fit to the experimental data presented in the same
format as in Fig. 2. Model represents Eqs.
1-8 fit to the data as indicated in text.
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To return to the potential role of the ascorbate that accumulated in
the conditioned perfusate (Fig. 3) in TBOP+ reduction in
the lungs, we obtained an estimate of the rate of ascorbate release by
the lungs and evaluated its contribution to TBOP+ reduction
in lungs as follows. Under the experimental conditions in the
conditioned perfusate samples, resulting in the data shown in
Fig. 3, the relevant reactions are reactions
b-d and Eqs. 5-8 reduce
to
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(9)
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(10)
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(11)
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To fit Eqs. 9-11 to the Fig. 3 data,
ko was set to the value estimated from the PSS
plus SOD data in Fig. 2, and k
was set to the standard
value of 12 µM
1 · min
1
(17). Thus the identifiable parameters were
k3
(µM
1 · min
1),
k
o
(µM
2 · min
1) and
[AH2]0 (µM), the concentration of ascorbate
in the conditioned perfusate sample before the addition of
TBOP+. The model fit to the TBOP+ data from the
conditioned perfusate samples was obtained by numerically solving
Eqs. 9-11 with the initial conditions
[TBOP+](t = 0) = [TBOP+]0, [AH2](t) = [AH2]0, and
[O
·](t) = 0. The estimated values of
k3 and k
o were 0.146 ± 0.1 (SE) µM
1 · min
1 and 1.03 ± 0.71 µM
2 · min
1, respectively.
Figure 8 shows the resulting estimate of
the concentration of ascorbate ([AH2]0) in
the conditioned perfusate, which increased approximately linearly with
recirculation time. The model estimated rate of 0.383 µM/min is
comparable to the value of 0.302 µM/min estimated by measuring the
ascorbate concentration (10), which provides additional
evidence for consistency in the model assumptions.

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|
Fig. 8.
Ascorbate concentration in conditioned perfusate vs.
recirculation time estimated by fitting Eqs.
9-11 to data exemplified in Fig. 3.
|
|
Knowing the rate of ascorbate accumulation in the conditioned medium,
the rate of ascorbate efflux from the lungs into the perfusate was
estimated with Eq. 12, which is the solution of a nested
version of Eqs. 1-8, descriptive of
ascorbate kinetics in TBOP-free recirculation experiments
|
(12)
|
where tc = Qc/F is
the pulmonary capillary transit time, estimated to be ~2.4 s under
the conditions of these experiments (14). The value of
kas was thus estimated to be 6.1 nmol/min.
To put the contributions of each of the three TBOP+
reduction mechanisms in prospective, model simulations of the pulmonary venous effluent TBOP+ concentration were generated by
solving Eqs. 1-8 with the appropriate initial and boundary conditions and with the parameters
ko, kred, kso, and k
o set to
values estimated from Fig. 7 fits to the lung data, with the additional
parameters k3 and kas set
to 0.146 µM
1 · min
1 and 6.1 nmol/min, respectively, estimated from the data in Fig. 3, and
k
set to the standard value 12 µM
1 · min
1 (17). The
objective was to determine how much each of the three reduction
mechanisms contributed to the experimental data by successively eliminating it from the simulations. For simulation 1 in
Fig. 9, all three reduction mechanisms
(reductase, ascorbate, and superoxide) were allowed to contribute to
the simulated TBOP+ reduction. Then, for simulation
2, the ascorbate contribution was eliminated. For simulation
3, the superoxide contribution was eliminated. For
simulation 4, both the ascorbate and superoxide contributions were eliminated, and for simulation 5, all
three reduction mechanisms (ascorbate, superoxide, and reductase) were eliminated by setting the appropriate parameters to zero (sham). Simulation 5 is a trivial result, but it is shown to
emphasize the range of possibilities. These simulations demonstrate the model explanation for the experimental observations, namely, that the
pulmonary endothelial TPMET dominated the pulmonary reduction of
TBOP+ and that the combined effect of ascorbate and
superoxide released from the lungs could account for no more than 13%
of the total TBOP+ reduction.

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|
Fig. 9.
, Pulmonary venous effluent
[TBOP+] (as a fraction of the initial
[TBOP+] added to the perfusate) vs. recirculation time
with no additions other than TBOP+. The lines are model
simulations generated with Eqs. 1-8. For
simulation 1, the contributions of reductase, superoxide,
and ascorbate to simulated TBOP+ reduction were set to the
values estimated as described in text. For simulation 2,
ascorbate contribution was eliminated. For simulation 3,
superoxide contribution was eliminated. For simulation 4,
both ascorbate and superoxide contributions were eliminated. For
simulation 5, the contributions of all 3 mechanisms were
eliminated.
|
|
 |
DISCUSSION |
The experimental results and kinetic model interpretation lead to
the conclusion that the major route in TBOP+ reduction
within the lungs is via an endothelial TPMET system. This relative
contribution of TPMET is emphasized by the model simulations shown in
Fig. 9, where it can be seen that the predicted contributions of
ascorbate and any superoxide that might be released by the lungs to the
pulmonary reduction of TBOP+ are very small compared with
that attributable to endothelial TPMET. Ascorbate and superoxide
contributions to TBOP+ were specifically addressed because
previous studies have demonstrated the release of ascorbate (1,
10) and, under some experimental conditions, superoxide
(10, 22, 32) by isolated perfused lungs. Although the
possibility that some other unidentified and very short-lived
TBOP+ reducing agent might contribute to TBOP+
reduction would be difficult to rule out entirely, it is difficult to
imagine what another candidate might be that would also be undetectable
in the immediately sampled conditioned perfusate. Neither NO nor
H2O2 are TBOP+ reductants. The
steady-state release of a reducing agent in sufficient quantity to
account for the reduction of the various thiazine dyes that have been
studied would have to be even greater than the normal rate of
O2 reduction by the lung (4, 7, 14).
Although the results indicate that the release of a reductant is not a
major contributor to TBOP+ reduction, the data in Fig. 3 do
demonstrate that a TBOP+ reductant accumulates in the
perfusate recirculating through the lungs. Its elimination by AO is
consistent with previous studies indicating ascorbate release by the
perfused lungs (1, 10). The estimated rate of release from
the data in Fig. 3 of ~4
nmol · min
1 · g wet wt
1 is
on the same order as that previously reported by Arad et al. (1) in perfused rat lungs (6 nmol · min
1 · g wet wt
1)
and by Bongard et al. (10) in perfused rabbit lungs (2 nmol · min
1 · g wet wt
1).
The rate of TBOP+ reduction in the lungs is 50 nmol · min
1 · g wet wt
1.
Although the conditioned perfusate had accumulated enough ascorbate by
the end of the perfusion period to reduce the added TBOP+
to a fraction comparable to that in the perfusate recirculating through
the lungs, the total amount released over the entire period was small
compared with the required reduction rate during that period. This
accounts for the small effect of adding AO to the recirculating
perfusate in the presence of TBOP+ as shown in Fig. 2.
The higher steady-state TBOP+ fraction in the presence of
SOD in Fig. 2 might be thought to suggest a significant role for superoxide in the pulmonary reduction of TBOP+. However,
the kinetic analysis apparently rules out that possibility. When both
TBOP+ reduction and TBOPH autooxidation rates are taken
into account, the effect of adding SOD can be almost completely
accounted for by its effect on the TBOPH autooxidation rate. Thus there
is no reason to believe that superoxide released by the lungs is a
significant TBOP+ reductant in lungs. This is explained
mechanistically by including superoxide as an autooxidation by-product
in the stoichiometric equations leading to the kinetic model. This
explanation for the SOD effect is analogous to those of Auclair et al.
(3) and Picker and Fridovich (36) for the
inhibitory effect of SOD on the aerobic reduction of nitro blue
tetrazolium (NBT) in the presence of NADPH and NADPH-cytochrome
P-450 reductase or NADH and phenazine methosulfate,
respectively. They determined that the SOD effect was on the superoxide
produced by autooxidation of the intermediate monoformazan formed in
the first step of the two-step NBT reduction to the diformazan. By
eliminating the superoxide formed in the autooxidation reaction, SOD
increases the autooxidation rate, i.e., the regeneration of NBT, and
thus causes inhibition of diformazan production. A minor difference for
TBOP is that TBOPH is the final two-electron reduction product, which
is autooxidizable, whereas the final two-electron NBT reduction product
diformazan is a stable end product. Although a superoxide release rate
is a model output, the estimated rate makes such a small contribution
to the model fit that its numerical value is useful only to show that
it cannot be an important contributor to TBOP+ pulmonary
reduction and not as an accurate measure of superoxide release by the lungs.
In the above analysis, the uncatalyzed superoxide dismutation rate
(k
) was set to the standard value of 12 µM
1 · min
1 (17),
which may or may not be an accurate estimate under the experimental
conditions of the present study. To evaluate the sensitivity of the
model predictions to this value, we varied the value of
k
from 3 to 36 µM
1 · min
1. The results of this
exercise indicated that the changes in k
were
compensated for almost exclusively by changes in
k
o, with little effect on the estimated values
of the other parameters or on the ability of the model to fit the data.
In other words, k
and
k
o are highly correlated with each other but not with the other model parameters. Similar analysis revealed the
robustness of the model fit and data interpretation to the ratio of
catalyzed to uncatalyzed superoxide dismutation rates (Kd) when Kd was >5.
Thus the lower bound Kd estimate of 2 × 102 from the data in Fig. 6 is quite sufficient support for
the assumption that in the presence of SOD, reaction b could
be considered unidirectional.
The estimated TBOP+ reduction rate was about two orders of
magnitude smaller than that previously estimated for oxidized TBO (TBO+) itself (4, 14), and the estimated TBOPH
autooxidation rate constant was about half of that estimated for
reduced TBO (14). The reasons for these differences are
not known, but at least two factors probably contribute. One is that
the TBO moieties in the polymer are no longer TBO because the primary
amine is changed to a complicated secondary amine (9).
This probably contributes to the shift in the absorbance spectrum in
the region of TBO maximum absorbance (from 626 nm for TBO+
to 590 nm for TBOP+). The high absorbance at the low
wavelengths is mainly due to the acrylamide polymer. Another likely
contributor to the differences in reduction rates is steric hindrance
due to the small redox-active moieties being attracted to the large polymer.
In summary, the experimental and kinetic model results are consistent
in pointing to the endothelial TPMET system as the major contributor to
TBOP+ reduction in lungs. The SOD effect was predominantly
on autooxidation rather than on reduction, and no long-lived
TBOP+ reductant was released into the perfusate in
sufficient quantity to contribute significantly to the
TBOP+ reduction. The basic experimental design with
TBOP+ as the extracellular electron acceptor and the
kinetic model for interpretation appear to provide tools for further
studies of pulmonary endothelial TPMET mechanisms and their responses to physiological and pathophysiological stresses.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood Grants
HL-24349 and HL65537 and the Department of Veterans Affairs.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
C. A. Dawson, Research Service 151, Zablocki VA Medical Center,
5000 W. National Ave., Milwaukee, WI 53295-1000 (E-mail:
cdawson{at}mcw.edu).
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.
Received 7 November 2000; accepted in final form 15 December 2000.
 |
REFERENCES |
1.
Arad, ID,
Forman HJ,
and
Fisher AB.
Ascorbate efflux from guinea pig and rat lungs.
J Lab Clin Med
96:
673-681,
1980[ISI][Medline].
2.
Arroyo, A,
Kagan VE,
Tyurin VA,
Burgess JR,
de Cabo R,
Navas P,
and
Villalba JM.
NADH and NADPH-dependent reduction of coenzyme Q at the plasma membrane.
Antioxid Redox Signal
2:
251-262,
2000[Medline].
3.
Auclair, C,
Voisin E,
and
Bandoun H.
Superoxide dismutase-inhibitable NBT and cytochrome c reduction as probe of superoxide anion production: a reappraisal.
In: Oxy Radicals and Their Scavenger Systems. Volume I: Molecular Aspects, edited by Cohen G,
and Greenwald RA.. New York: Elsevier Science, 1983, p. 312-315.
4.
Audi, SH,
Olson LE,
Bongard RD,
Roerig DL,
Schulte ML,
and
Dawson CA.
Toluidine blue O and methylene blue as endothelial redox probes in the intact lung.
Am J Physiol Heart Circ Physiol
278:
H137-H150,
2000[Abstract/Free Full Text].
5.
Baker, MA,
and
Lawen A.
Plasma membrane NADH-oxidoreductase system: a critical review of the structure and functional data.
Antioxid Redox Signal
2:
197-212,
2000[Medline].
6.
Berridge, MV,
and
Tan AS.
High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane, cell surface, and serum NADH-oxidases.
Antioxid Redox Signal
2:
231-242,
2000[Medline].
7.
Bongard, RD,
Krenz GS,
Linehan JH,
Roerig DL,
Merker MP,
Widell JL,
and
Dawson CA.
Reduction and accumulation of methylene blue by the lungs.
J Appl Physiol
77:
1480-1491,
1994[Abstract/Free Full Text].
8.
Bongard, RD,
Merker MP,
Daum JM,
and
Dawson CA.
Quinone reduction by endothelial cells: potential mechanism for regulating redox status of low density lipoproteins (LDL) (Abstract).
FASEB J
13:
A185,
1999[ISI].
9.
Bongard, RD,
Merker MP,
Shundo R,
Okamoto Y,
Roerig DL,
Linehan JH,
and
Dawson CA.
Reduction of thiazine dyes by bovine pulmonary arterial endothelial cells in culture.
Am J Physiol Lung Cell Mol Physiol
269:
L78-L84,
1995[Abstract/Free Full Text].
10.
Bongard, RD,
Roerig DL,
Johnston MR,
and
Dawson CA.
Perfusate cytochrome c reduction in isolated rabbit lungs.
J Appl Physiol
71:
1705-1713,
1991[Abstract/Free Full Text].
11.
Chueh, PJ.
Cell membrane redox systems and transformation.
Antioxid Redox Signal
2:
177-188,
2000[Medline].
12.
Crane, F,
Sun LL,
Barr R,
and
Low H.
Electron and proton transport across the plasma membrane.
J Bioenerg Biomembr
23:
733-803,
1991.
13.
Cross, AR.
The inhibitory effects of iodium compounds on the superoxide generating system of neutrophils and their failure to inhibit diaphorase activity.
Biochem Pharmacol
36:
489-493,
1987[ISI][Medline].
14.
Dawson, CA,
Audi SH,
Bongard RD,
Okamoto Y,
Olson L,
and
Merker MP.
Transport and reaction at endothelial plasmalemma: distinguishing intra- from extracellular events.
Ann Biomed Eng
28:
1010-1018,
2000[ISI][Medline].
15.
Dawson, CD,
Bongard RD,
Merker MP,
Olson LE,
and
Linehan JH.
Pulmonary endothelium reduces copper and ubiquinone: implications for atherosclerosis (Abstract).
J Vasc Res
35:
61,
1998[ISI].
16.
Dixon, M,
and
Webb EC.
Enzymes (3rd ed.). San Francisco: Academic, 1979, p. 483.
17.
Fridovich, I.
Superoxide dismutases.
Annu Rev Biochem
44:
147-159,
1975[ISI][Medline].
18.
Garner, B,
vanReyk D,
Dean RT,
and
Jessup W.
Direct copper reduction by macrophages.
J Biol Chem
272:
6927-6935,
1997[Abstract/Free Full Text].
19.
Giulivi, C,
and
Cadenas E.
Extracellular activation of fluorinated azriridinylbenzoquinone in HT29 cells EPR studies.
Chem Biol Interact
113:
191-204,
1998[ISI][Medline].
20.
Goldenberg, H,
Landershamer H,
and
Laggner H.
Functions of vitamin C as a mediator of transmembrane electron transport in blood cells and related cell culture models.
Antioxid Redox Signal
2:
189-196,
2000[Medline].
21.
Kaul, N,
Choi J,
and
Forman JH.
Transmembrane redox signaling activities NF-kappa B in macrophages.
Free Radic Biol Med
24:
202-207,
1998[ISI][Medline].
22.
Kennedy, TP,
Rao NV,
Hopkins C,
Pennington L,
Tolley E,
and
Hoidal JR.
Role of reactive oxygen species in reperfusion injury of the rabbit lung.
J Clin Invest
83:
1326-1335,
1989[ISI][Medline].
23.
Marshall, C,
Mamary AJ,
Verhoeven AJ,
and
Marshall BE.
Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.
Am J Respir Cell Mol Biol
15:
633-644,
1996[Abstract].
24.
McCord, JM
and Fridovich I. The utility of superoxide dismutase in studying free radical reactions. II: the mechanism of the mediation of cytochrome c reduction by a variety of electron carriers.
J Biol Chem
245:
1374-1377,
1970[Abstract/Free Full Text].
25.
Merker, MP,
Audi SH,
Brantmeier BM,
Nithipatikom K,
Goldman RS,
Roerig DL,
and
Dawson CA.
Proline in vasoactive peptides: consequences for peptide hydrolysis in the lung.
Am J Physiol Lung Cell Mol Physiol
276:
L341-L350,
1999[Abstract/Free Full Text].
26.
Merker, MP,
Bongard RD,
Linehan JH,
Okamoto Y,
Vyparachticky D,
Brantmeier BM,
Roerig DL,
and
Dawson CA.
Pulmonary endothelial thiazine uptake: separation of cell surface reduction from intracellular reoxidation.
Am J Physiol Lung Cell Mol Physiol
272:
L673-L680,
1997[Abstract/Free Full Text].
27.
Merker, MP,
Olson LE,
Bongard RD,
Patel MK,
Linehan JH,
and
Dawson CA.
Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells.
Am J Physiol Lung Cell Mol Physiol
274:
L685-L693,
1998[Abstract/Free Full Text].
28.
Mohazzab, KM,
Kaminski PM,
Agarwal R,
and
Wolin MS.
Potential role of a membrane-bound NADH oxidoreductase in nitric oxide release and arterial relaxation to nitroprusside.
Circ Res
84:
220-228,
1999[Abstract/Free Full Text].
29.
Morre, DJ,
Sun E,
Geilen C,
Wu LY,
De Cabo R,
Krasagakis K,
Orfanos CE,
and
Morre DM.
Capsaicin inhibits plasma membrane NADH oxidase and growth of human and mouse melanoma lines
Eur J Cancer
32A:
1995-2003,
1996.
30.
Nakamura, M,
and
Hayashi T.
One- and two-electron reduction of quinines by rat liver subcellular fractions.
J Biochem (Tokyo)
115:
1141-1147,
1994[Abstract].
31.
Navarro, F,
Navas P,
Burgress JR,
Bello RI,
De Cabo R,
Arroyo A,
and
Villalba JM.
Vitamin E and selenium deficiency induces expression of the ubiquinone-dependent antioxidant system at the plasma membrane.
FASEB J
12:
1665-1673,
1998[Abstract/Free Full Text].
32.
Nozik-Grayck, E,
Piantadosi CA,
Van Adelsberg J,
Alper JL,
and
Huang YT.
Protection of perfused lung from oxidant injury by inhibitors of anion exchange.
Am J Physiol Lung Cell Mol Physiol
273:
L296-L304,
1997[Abstract/Free Full Text].
33.
Nunez, MT,
Alvarez X,
Smith M,
Tapia V,
and
Glass J.
Role of redox system on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells.
Am J Physiol Cell Physiol
267:
C1582-C1588,
1994[Abstract/Free Full Text].
34.
Olson, LE,
Merker MP,
Patel MK,
Bongard RD,
Daum JM,
Johns RA,
and
Dawson CA.
Cyanide increases reduction but decreases sequestration of methylene blue by endothelial cells.
Ann Biomed Eng
28:
85-93,
2000[ISI][Medline].
35.
Pagano, PJ,
Ito Y,
Tornheim K,
Gallop PM,
Tauber AI,
and
Cohen RA.
An NADPH oxidase superoxide-generating system in the rabbit aorta.
Am J Physiol Heart Circ Physiol
268:
H2274-H2280,
1995[Abstract/Free Full Text].
36.
Picker, SD,
and
Fridovich I.
On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium.
Arch Biochem Biophys
228:
155-158,
1984[ISI][Medline].
37.
Rajagopalan, S,
Kurz S,
Munzel T,
Tarpey M,
Freeman BA,
Griendling KK,
and
Harrison DG.
Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone.
J Clin Invest
97:
1916-1923,
1996[Abstract/Free Full Text].
38.
Ross, R.
The pathogenesis of atherosclerosis: a perspective for the 1990's.
Nature
362:
801-809,
1993[ISI][Medline].
39.
Smalley, DM,
Hogg N,
Kalyanaraman B,
and
Pritchard KA, Jr.
Endothelial cells prevent accumulation of lipid hydroperoxides in low density lipoproteins.
Arterioscler Thromb Vasc Biol
17:
3469-3475,
1997[Abstract/Free Full Text].
40.
Smith, A.
Links between cell-surface events involving redox-active copper and gene regulation in the hemopexin heme transport system.
Antioxid Redox Signal
2:
157-176,
2000[Medline].
41.
Wollman, EE,
Kahan A,
and
Fradelizi D.
Detection of membrane associated thioredoxin on human cell lines.
Biochem Biophys Res Commun
230:
602-606,
1997[ISI][Medline].
Am J Physiol Lung Cell Mol Physiol 280(6):L1290-L1299