Ascorbate-mediated transplasma membrane electron transport in
pulmonary arterial endothelial cells
Marilyn P.
Merker1,2,4,
Lars E.
Olson2,
Robert D.
Bongard3,
Meha K.
Patel2,
John H.
Linehan2,3,4, and
Christopher
A.
Dawson2,3,4
Departments of 1 Anesthesiology
and Pharmacology and
3 Physiology, Medical College
of Wisconsin, Milwaukee 53226;
2 Biomedical Engineering
Department, Marquette University, Milwaukee 53201-1881; and
4 Research Service, Zablocki
Veterans Affairs Medical Center, Milwaukee, Wisconsin
53295
 |
ABSTRACT |
Pulmonary
endothelial cells are capable of reducing certain electron acceptors at
the luminal plasma membrane surface. Motivation for studying this
phenomenon comes in part from the expectation that it may be important
both as an endothelial antioxidant defense mechanism and in redox
cycling of toxic free radicals. Pulmonary arterial endothelial cells in
culture reduce the oxidized forms of thiazine compounds that have been
used as electron acceptor probes for studying the mechanisms of
transplasma membrane electron transport. However, they reduce another
commonly studied electron acceptor, ferricyanide, only very slowly by
comparison. In the present study, we examined the influence of
ascorbate [ascorbic acid (AA)] and dehydroascorbate
[dehydroascorbic acid (DHAA)] on the ferricyanide and
thiazine reductase activities of the bovine pulmonary arterial
endothelial cell surface. The endothelial cells were grown on
microcarrier beads so that the reduction of ferricyanide and methylene
blue could be studied colorimetrically in spectrophotometer cuvettes
and in flow-through cell columns. The ferricyanide reductase activity
could be increased 80-fold by adding DHAA to the medium, with virtually
no effect on methylene blue reduction. The DHAA effect persisted after
the DHAA was removed from the medium. AA also stimulated the
ferricyanide reductase activity but was less potent, and the relative
potencies of AA and DHAA correlated with their relative rates of uptake
by the cells. The results are consistent with the hypothesis that AA is
an intracellular electron donor for an endothelial plasma membrane
ferricyanide reductase and that the stimulatory effect of DHAA is the
result of increasing intracellular AA. Adding sufficient DHAA to
markedly increase extracellular ferricyanide reduction had little
effect on the plasma membrane methylene blue reductase activity,
suggesting that pulmonary arterial endothelial cells have at least two
separate transplasma membrane electron transport systems.
ferricyanide; cytochrome c; methylene blue; dehydroascorbic acid; endothelial cell column; ascorbic
acid transport; dehydroascorbic acid transport
 |
INTRODUCTION |
PULMONARY ENDOTHELIAL CELLS are capable of reducing
electron acceptors at the luminal plasma membrane surface via
transplasma membrane electron transport (3, 14). The role of such a
system in normal vascular biology has not yet been identified. However, the existence of the high-capacity reducing system in direct contact with the blood suggests an impact on the redox status of blood constituents, with implications with regard to the physiological role
of the endothelium in antioxidant defense. It is also a mechanism whereby the lungs could affect the systemic circulation by modifying the redox status of blood constituents entering the arterial system. It
has been shown to be important in the pharmacokinetics of certain redox-sensitive drugs (2, 14), and it is likely to be involved in the
mechanism of action and disposition of redox-cycling toxins and
chemotherapeutic agents.
Transplasma membrane electron transport systems are found in various
mammalian cell types (8, 10, 15, 26). Ferricyanide is commonly used as
an electron acceptor in studies of transplasma membrane electron
transport because neither ferricyanide nor its reduced form,
ferrocyanide, enters the cells. Thus any reduction can be attributed
solely to the cell surface. Ferricyanide reductase systems are present
in the plasma membranes of hepatocytes (8), erythrocytes (1, 12, 17,
20, 22), and other cells (6, 8), and they may be ubiquitous. However,
ferricyanide has been found to be only very slowly reduced by pulmonary
arterial endothelial cells in culture in comparison with several other
electron acceptors reduced on the cell surface (3).
In the process of attempting to identify the mechanisms involved in
endothelial transplasma membrane electron transport, we have made the
observation that the relatively inactive ferricyanide reductase
activity can be stimulated by supplying dehydroascorbate [dehydroascorbic acid (DHAA)] in the cell medium. The
results are consistent with the hypothesis that ascorbate
[ascorbic acid (AA)] is an intracellular electron donor for
this reductase and that the stimulatory effect of DHAA is the result of
increasing the intracellular AA concentration. Adding sufficient DHAA
to markedly increase extracellular ferricyanide reduction had no effect
on plasma membrane thiazine reductase activity, suggesting that there
are at least two separate transplasma membrane electron transport
systems in pulmonary arterial endothelial cells.
 |
METHODS |
Reagents.
Potassium ferricyanide, L-AA,
ascorbate oxidase (AO; A-0157), HEPES, lactate dehydrogenase (LDH) kit
340-LD and enzyme control 2E, ferricytochrome
c (from horse heart), Triton X-100,
mouse anti-
-actin, tetramethylrhodamine isothiocyanate-labeled goat anti-mouse IgG, and FITC-dextran (2 × 106 g/mol) were purchased from
Sigma (St. Louis, MO).
Dehydro-L-(+)-AA dimer was also
obtained from Sigma. Because the dimer dissociates on hydration, the
units reported are for the monomeric DHAA. Experiments with DHAA were
initiated within 2 min of dissolving the DHAA in the medium.
D-Glucose was from Fisher
Scientific (Fair Lawn, NJ), and sodium hydrosulfite was from
Mallinckrodt Chemical Works (St. Louis, MO). Methylene blue chloride
was obtained from E. M. Science (Gibbstown, NJ).
[14C]AA was from NEN
Life Science Products (Boston, MA). Hanks' balanced salt solution
(HBSS), trypsin, penicillin-streptomycin, and RPMI 1640 tissue culture
medium were from GIBCO (Grand Island,
NY). Fetal bovine serum was from Hyclone Laboratories (Logan, UT), and
Biosilon beads were from A/S Nunc (Roskilde, Denmark). Type CLS2
collagenase was obtained from Worthington Biochemicals (Freehold, NJ),
and diiodoindocarbocyanine-acetylated low-density lipoprotein was
purchased from Biomedical Technologies (Stoughton, MA). Protein determinations were performed using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Endothelial cell culture.
The bovine pulmonary endothelial cells used in this study were obtained
from two sources. One source was calf pulmonary artery obtained from a
local meat-processing plant as previously described (14). The second
source was cell line CPAE (CCL-209) from the American Type Culture
Collection (Rockville, MD). The cells were cultured in T75 tissue
culture flasks maintained at 37°C in RPMI 1640 (for the cells
obtained locally) or minimal essential Eagle's medium (for the CPAE
cells). Both media contained 10% heat-inactivated fetal bovine serum,
100 U/ml of penicillin, 100 µg/ml of streptomycin, and 30 mg/ml of L-glutamine.
The endothelial cell characteristics of the cultures were confirmed by
cobblestone morphology observed by phase-contrast microscopy. Cells
from either source were >95% endothelial cells as determined by
diiodoindocarbocyanine-acetylated low-density lipoprotein uptake
observed by fluorescence microscopy and immunofluorescence staining for
-actin.
To obtain a large cell-surface area for experiments carried out in
spectrophotometric cuvettes or in cell columns, the cells grown to
confluence in T75 tissue culture flasks were detached from the flasks
by treatment with trypsin (0.05% wt/vol) in HBSS, and ~5 × 106 cells were seeded onto 3 g
(dry wt) of gelatin-coated (2% vol/wt) Biosilon beads (mean diameter
230 µm; A/S Nunc). The cells on the beads were cultured in biological
stirrers (Techne, Princeton, NJ) in the medium described above.
Stirring (60 rpm) was intermittent (2 min on, 30 min off) during cell
attachment (~18-24 h) and continuous thereafter. The cells were
grown to confluence on the beads as determined by observation with
phase-contrast microscopy. The two cell types provided qualitatively
similar results in the experiments to be described. However, the CPAE
cell line tended to have a smaller total protein and LDH activity per
unit surface area and a thinner profile on the beads than the cells
grown from those initially harvested in our laboratory.
Reduction of ferricyanide and cytochrome c and
uptake of methylene blue by cells in cuvettes.
The general procedure used has been described previously (3, 14). About
0.5 ml of the cell-coated beads was transferred to 10 × 10 × 55-mm polystyrene spectrophotometer cuvettes
(Sarstedt, Newton, NC) and washed free of culture medium using three
consecutive 3-ml volumes of HBSS containing 10 mM HEPES and 5.5 mM
glucose at pH 7.4 (referred to subsequently as H-H). This procedure and subsequent experiments were carried out at room temperature,
~24°C. After the third wash, 2 ml of H-H solution containing
ferricyanide (300 µM), ferricytochrome
c (75 µM), or methylene blue (10 µM), each with or without DHAA (0-300 µM), were added to the
cuvettes. Once the cell-coated beads had settled to the bottom (~25
s), the cuvettes were sealed, and the initial absorbance at 421, 550, or 611 nm was recorded for samples containing ferricyanide, cytochrome c, or methylene blue, respectively,
using a Gilford Response spectrophotometer (Ciba Corning Diagnostics,
Norwood, MA). The reduced form of ferricyanide (ferrocyanide) and
reduced methylene blue do not absorb at these wavelengths. Therefore,
the concentrations of ferricyanide and the oxidized form of methylene
blue (MB+) were determined by
comparing the recorded absorbance values with standard curves of known
concentration prepared the day of the experiment. Reduced cytochrome
c concentrations were determined using
an extinction coefficient of 2.1 × 107
cm2/mol. The cuvette contents were
gently mixed using a Nutator mixer (Clay Adams Division of Becton
Dickinson, Parsippany, NJ), and at 2.5-min intervals, the cuvettes were
placed in the spectrophotometer where the cell-coated beads were
allowed to settle. Absorbance was measured at the appropriate
wavelength(s), and the cuvettes were returned to the mixer. In some
experiments, the washed cell-coated beads were mixed with H-H
containing either DHAA (100 µM) or AA (100 µM) for a period of 15 min and then washed a second time using fresh H-H before the addition
of 2 ml of H-H containing either ferricyanide (300 µM) or
MB+ (10 µM). The total
concentration of ferricyanide+ferrocyanide in the H-H bathing the cells
was determined after the experimental period by oxidizing any
ferrocyanide present with 0.1 ml of 3% H2O2
and 0.1 ml of 70% HClO4/ml of
H-H. Then, the difference in ferricyanide concentrations before and
after the oxidation was the measure of the ferrocyanide present before
the oxidation. Similarly, total cytochrome
c or total methylene blue
(oxidized+reduced) was determined by oxidizing any of the reduced forms
present by the addition of 0.025 ml of ferricyanide (10 mM)/ml of H-H.
In each cuvette experiment, the experimental and various control
cuvettes were run simultaneously with cells grown in the same flask to
control for cell source and other possible cell culture effects on the
reductase activity. For each redox indicator and experimental medium
composition, the number of cuvettes studied and the figures showing
examples of the data are as follows: ferricyanide alone, 10 cuvettes, see Figs. 1-3; ferricyanide+DHAA, 15 cuvettes, see Figs.
1-3, 5, and 6; ferricyanide after DHAA pretreatment, 6 cuvettes,
see Figs. 1 and 2; ferricyanide after AA pretreatment, 2 cuvettes, see
Fig. 2; ferricyanide+AO, 1 cuvette, see Fig. 5; ferricyanide+DHAA+AO, 3 cuvettes, see Fig. 5;
ferricyanide+ AA+AO, 2 cuvettes, see Fig. 5; cytochrome
c alone, 2 cuvettes, see Fig. 7;
cytochrome c+DHAA, 2 cuvettes, see
Fig. 7; methylene blue alone, 2 cuvettes, see Fig. 8; methylene
blue+DHAA, 1 cuvette, see Fig. 8; and methylene blue after pretreatment
with DHAA, 4 cuvettes, see Fig. 8. In each case, the examples shown are
representative of the results of all experiments performed.
To determine the efficacy of ferricytochrome
c reduction as an in situ AA assay, 20 ml of H-H containing ferricytochrome c (75 µM) were placed in a beaker. AA (0.5 mM) was directly infused into the stirred cytochrome c solution
at a rate of 6.8 µl/min. The cytochrome
c solution absorbance (550 nm) was
measured over a period of 100 min. A control was performed in a like
manner using AA-free H-H infusion.
Reduction of ferricyanide and
MB+ by the cells
in the cell column.
The flow-through column containing endothelial cell-covered beads has
been described previously (16). To prepare the cell column, ~1 ml of
cell-covered beads was removed from the culture flask and washed three
times with fresh H-H. The beads were placed in a chromatographic column
(5.0 × 0.7 cm; Econo-Column, Bio-Rad) with a plastic frit
(Bel-Art, Pequannock, NJ) and a 100-µm nylon macrofiltration mesh
screen (Cole-Parmer, Niles, IL) at the bottom. A plunger was placed on
the top of the column touching the top layer of beads. The cell column
was perfused with H-H at flows ranging from 0.3 to 1.0 ml/min with a
peristaltic pump (Buchler Polystaltic Pump; Hakke/Sissons Instruments,
Paramus, NJ). Upstream from the cell column, a Rheodyne Teflon loop
(100 µl) injection valve (Rainin Instruments, Woburn, MA) was
included in the perfusion system so that a bolus of H-H containing
FITC-dextran (0.75 µM) and either ferricyanide (200 µM) or
MB+ (28 µM) could be introduced
at the desired time without changing flow or pressure. The effluent dye
concentrations were measured on-line downstream from the column at 490 nm (FITC-dextran) and at either 420 (ferricyanide) or 590 nm
(MB+) using the custom-built
optical detector described previously (16). The column experiments were
also carried out at room temperature. In the cell column experiments,
because of the ability to make repeated measurements, each cell column
served as its own control.
Four experimental protocols were followed using the cell columns. The
first was designed to determine the effect of DHAA on ferricyanide
reduction. Bolus injections containing FITC-dextran and ferricyanide
were made with flow rate set at 0.3, 0.7, and 1.0 ml/min (column
transit times of ~60, 26, and 18 s, respectively). The cells were
then perfused with H-H containing 200 µM DHAA for 30 min at 1.0 ml/min. The perfusion medium was then changed back to H-H without DHAA
for 10 min, and the boluses were injected at the same three flow rates
as before the addition of DHAA. The second protocol was identical to
the first except that the boluses contained
MB+ instead of ferricyanide. The
third protocol was used to determine the time course of the DHAA effect
on ferricyanide reduction. Bolus injections of FITC-dextran and
ferricyanide were made every 5 min, with cell column flow set at 1.0 ml/min. Three injections were made during perfusion with H-H alone. The
cell column was then perfused with H-H containing 200 µM DHAA for 56 min. Bolus injections were made 15 min after the perfusate change and
at 5-min intervals thereafter. At the end of the DHAA perfusion, the
perfusate was changed back to DHAA-free H-H. After 10 min, bolus
injections were again made at 5-min intervals for an additional 25 min.
Each change in medium composition was over 95% complete in <4 min.
The fourth protocol was identical to the third except that the boluses
contained MB+ instead of
ferricyanide. The fractions of MB+
and ferricyanide reduced during passage of the bolus through the column
were calculated by first dividing the respective indicator concentrations by their respective injected amounts (see Figs. 9 and
10). Then the areas under the fractional concentration curves were
measured from the appearance time to the peak of the FITC-dextran curve. The difference in these areas under the
MB+ or ferricyanide curves and
this area under the FITC-dextran curve divided by this area under the
FITC-dextran curve was the fraction of
MB+ or ferricyanide reduced.
Cell uptake of [14C]AA and
[14C]DHAA.
The cell-coated beads were washed free of culture medium using H-H and
dispensed (~0.25 ml) into six 1.5-ml Eppendorf tubes. A 1-ml volume
of H-H containing
[14C]AA (35 µM;
specific activity 10 mCi/mmol) was added to the cell-coated beads in
five of the tubes, and 1 ml of H-H was added to the cells in the
remaining tube. The tubes were capped and gently mixed at room
temperature using a Nutator mixer. Uptake was terminated at 5, 10, 15, 20, or 30 min by washing the cells with four consecutive 1.25-ml
volumes of fresh H-H and then solubilizing the cells with 0.75 ml of
0.5 N NaOH containing 2% Triton X-100. The samples were mixed
overnight, and radioactivity of the solubilized cells (0.5 ml) and
final-wash H-H (0.5 ml) from each sample was measured in 10 ml of
Bio-Safe II liquid scintillation cocktail (Research Products
International, Mount Prospect, IL) using liquid scintillation spectrometry (Packard TriCarb model 3330 liquid scintillation spectrometer, Downers Grove, IL). Counting efficiencies were determined by the addition of
[14C]toluene (0.01 ml;
specific activity 4.4 × 105
dpm/g) to each of the vials, which were then counted a second time. The
balance of the solubilized cell samples was assayed for protein. The
uptake of 14C from
[14C]AA expressed as
nanomoles per minute per milligram of protein was calculated from the
disintegrations per minute and the
[14C]AA specific
activity. The uptake of 14C from
[14C]DHAA was measured
in a similar manner after generation of
[14C]DHAA by reacting
[14C]AA with AO (2 U/ml) for 5 min. The 14C uptake
was measured in 36 cuvettes: 24 with
[14C]DHAA and 12 with
[14C]AA added to the
medium.
Assessment of the presence of AA in
cell-conditioned medium.
Cell-coated beads in three cuvettes were washed free of culture medium,
and 2 ml of H-H containing DHAA (100 µM) and ferricyanide (300 µM)
were added to one cuvette, and 2 ml of H-H containing only DHAA (100 µM) were added to the other two cuvettes. The three cuvettes were
sealed, and the absorbance at 421 nm was recorded over time as
described in Reduction of ferricyanide and cytochrome c and uptake of methylene blue by cells in cuvettes.
The experimental period was halted when the absorbance of the
ferricyanide-containing sample approached zero, and further mixing
resulted in no additional decrease in absorbance (100 min). The
ferricyanide-reducing capacity of the H-H contained in the
ferricyanide-free cuvettes after the experimental period was determined
by transferring a 1.7-ml volume of the cell-conditioned medium to a
clean cuvette containing ferricyanide (300 µM). The absorbance
between 350 and 450 nm was recorded in 2-nm intervals, and the
resulting spectrum was compared with the spectrum of a ferricyanide
solution (300 µM) prepared using fresh H-H. The H-H contained in the
remaining ferricyanide-free cuvette was transferred to a quartz
cuvette, and absorbance between 200 and 300 nm was recorded in 2-nm
intervals using fresh H-H as the blank.
LDH and total protein determinations.
The LDH activity of the cells and medium was measured as an index of
cell viability for comparison between experimental conditions (14).
After the experimental period, the medium bathing the cells was
aspirated and the cells were lysed on ice in 2.5 ml of cold deionized
water using a Virsonic cell disruptor (model 16-850; Virsonic,
Gardiner, NY) set to administer three 10-s exposures at 35% maximum
intensity to each sample. A volume of each lysate was diluted 1:5 with
0.1 M sodium phosphate buffer (pH 7.5), and the diluted lysate and
aspirated medium were assayed in duplicate for LDH using the
spectrophotometric method of Wroblewski and LaDue (24). Paired control
LDH samples (Sigma enzyme control 2E) were run the day of each
experiment to ensure assay reliability. The amounts of LDH are given in
Sigma units, where one unit of LDH results in a decrease in optical
absorbance (340 nm) of 0.001/min at 25°C in a 3-ml reaction volume
with a 1-cm light path. The protein concentration of each sample of
cell-bathing medium and cell lysate (diluted 1:20) was determined using
the Bio-Rad protein assay, which is based on the Bradford dye-binding
procedure (4). Paired samples were compared with standard curves
prepared the day of each experiment using known concentrations of BSA.
Because the typical experimental protocol used in this study involved changing the medium or the flow-through system, the cell-to-medium LDH
ratio was not necessarily highly sensitive for comparisons between
conditions. Therefore, the LDH-to-total cell protein ratio is also
reported for assessing the possibility that different experimental
conditions might have had different effects on cell viability. The LDH
found in the medium in the cuvettes as a fraction of the total
(cell+medium) at the time of measurement averaged 3.8 ± 3.7% (mean ± SD), with no systematic differences detected among study
conditions.
 |
RESULTS |
Figure 1 is an example of the initial
observation that including DHAA in the medium surrounding the
endothelial cells stimulated reduction of ferricyanide. Because DHAA
itself does not reduce ferricyanide, the reduction of the ferricyanide
under these conditions was cell mediated. The observation that nearly
all 600 nmol of the ferricyanide added to the medium were reduced,
although only 200 nmol DHAA were present (i.e., the capacity to reduce
only 400 nmol of ferricyanide if each DHAA resulted in only one AA), was suggestive of a recycling process. Figure 1 also shows that the
stimulatory effect of DHAA was maintained even after the DHAA had been
removed from the medium.

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Fig. 1.
Effects of dehydroascorbate [dehydroascorbic acid (DHAA)]
on ferricyanide reduction by endothelial cells. Data from 2 cuvettes
are shown. In 1 cuvette (solid symbols), medium added at
time 0 contained ferricyanide (300 µM) and no DHAA. In other cuvette (open symbols), medium added at
time 0 contained ferricyanide (300 µm) and DHAA (100 µM). At 90 min, cells in both cuvettes were
washed, and fresh medium containing 300 µM ferricyanide but no DHAA
was added to both cuvettes. Thus, in cuvette designated by open
symbols, ferricyanide reduction was measured while DHAA was present in
medium ( ) and then again after DHAA had been removed ( ); this
cuvette contained 69.4 cm2 of
endothelial surface, 1.22 mg of protein, and 2.59 lactate dehydrogenase
(LDH) units/µg protein. In cuvette designated by solid symbols,
ferricyanide reduction was measured with no exposure of cells to DHAA;
this cuvette contained 68.1 cm2 of
endothelial surface, 1.29 mg of protein, and 2.44 LDH units/µg
protein.
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To determine whether AA itself was capable of stimulating ferricyanide
reduction, it was necessary to expose the cells to AA and then to
remove the AA from the medium before adding the ferricyanide. This is
because, in contrast to DHAA, AA directly reduces ferricyanide. Thus,
for the experiments depicted in Fig. 2, the
cells were first incubated for 15 min with AA, with AA+AO, or with
DHAA. Then the medium was replaced with H-H containing ferricyanide but
no other additives. In addition, a cuvette containing both DHAA and
ferricyanide was included for comparison. Pretreatment with DHAA or AA
stimulated ferricyanide reductase activity. However, AA was less
effective than DHAA unless AO had been present in the medium. This
effect of AO was presumably the result of AA conversion to DHAA.

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Fig. 2.
Effects of pretreatment of cells with DHAA or ascorbate [ascorbic
acid (AA)] on ferricyanide reduction by endothelial cells. Cells
were pretreated by including following additives in medium for 15 min
before time 0: DHAA (100 µM); AA
(100 µM); and ascorbate oxidase (AO; 4 units) plus AA (100 µM). Two
cuvettes received no pretreatment (none). Then cells were washed, and
fresh medium containing ferricyanide (300 µM) was added. Fresh medium
contained no other additives except where indicated (* DHAA was
included in medium after wash). Thus, except for 1 cuvette (*), various
additions were present in medium before but not during measurement of
ferricyanide reduction. Five cuvettes contained (mean ± SD) 92.9 ± 2.8 cm2 of endothelial
surface, 3.75 ± 0.12 mg of protein, and 3.37 ± 0.14 LDH
units/µg protein.
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The stimulatory effect of DHAA was found to be DHAA concentration
dependent and saturable (Fig. 3). By
fitting a hyperbolic function of Michaelis-Menten form to the data in
Fig. 3 (solid line), the maximum rate of the DHAA-stimulated reduction
of a 300 µM ferricyanide solution was estimated by nonlinear
regression analysis to be 8.2 nmol · min
1 · mg
protein
1. The concentration
of DHAA that produced a rate equal to one-half the extrapolated maximum
under the conditions of these experiments was estimated to be
0.10 mM.

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Fig. 3.
Rate of reduction of ferricyanide (300 µM) as a function of DHAA
concentration of medium. Five cuvettes contained (mean ± SD) 89.5 ± 1.5 cm2 of endothelial
surface, 1.68 ± 0.04 mg of protein, and 2.70 ± 0.23 LDH
units/µg protein.
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To determine whether DHAA and AA were in fact taken up by the cells,
[14C]DHAA or
[14C]AA was added to
the cells under the same conditions in which the ferricyanide reduction
was measured. The 14C accumulated
in the cells considerably faster when it was added as
[14C]DHAA than as
[14C]AA, consistent
with the relative potencies of DHAA and AA in stimulating ferricyanide
reductase (Fig. 4).

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Fig. 4.
Average (±SE) amount of 14C in
endothelial cells vs. time after adding
[14C]AA
(n = 2 cuvettes for each point) or
[14C]DHAA
(n = 4 cuvettes for each
point). Thirty-six cuvettes contained (mean ± SD) 48.3 ± 0.7 cm2 of endothelial surface and
1.36 ± 0.22 mg of protein.
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Figure 5 provides one piece of evidence that the release
of AA into the medium was not responsible for the DHAA effect on ferricyanide reduction. When AO was added to oxidize any AA that might
have been released into the medium, the stimulatory effect of DHAA on
ferricyanide reduction was not diminished. Controls included in this
experiment demonstrated that the results with DHAA could be reproduced
using AA if AO was included in the medium, thereby indicating that a
relevant concentration of AO had been used. Figure 5 also shows that AO
itself had no effect on ferricyanide reduction.

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Fig. 5.
Effects of AO on DHAA stimulation of ferricyanide reduction by
endothelial cells. At time 0, medium
containing ferricyanide (300 µM) was added to each cuvette. For
control, no other additions were made. Additions to other cuvettes: AO
(4 units); AO (4 units) plus AA (100 µM); DHAA (100 µM); and AO (4 units) plus DHAA (100 µM). Five cuvettes contained (mean ± SD)
90.1 ± 1.0 cm2 of endothelial
surface, 1.73 ± 0.06 mg of protein, and 2.36 ± 0.06 LDH
units/µg protein.
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Additional experiments were also performed to determine whether AA
released from the cells into the medium might make a significant contribution to the DHAA-stimulated ferricyanide reduction. As one
approach to this question, we found that the conditioned medium removed
from the DHAA-treated cells did not reduce ferricyanide (Fig.
6). In another approach, ferricytochrome
c, which is directly reduced by AA but
not by the cells within the relevant time frame, was added to the cells
in the presence of DHAA as a colorimetric assay for AA within the
cell-bathing medium (Fig. 7). There was virtually no reduction of ferricytochrome
c in comparison with the amount of AA
that would have been necessary to account for the reduction of
ferricyanide in the presence of the DHAA-stimulated cells. As a final
approach, conditioning the DHAA-containing medium by exposure to the
cells did not increase the ultraviolet (UV) absorption by the medium in
the 270-nm region of the spectrum, also indicating that little AA was
released from the cells in comparison with that required to account for
the ferricyanide reduction.

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Fig. 6.
Comparison of absorption spectrum of fresh medium containing DHAA (100 µM) plus ferricyanide (300 µM) with spectrum of medium that
contained DHAA during conditioning by cells but had ferricyanide added
after removal from cells and with spectrum of medium that contained
both DHAA and ferricyanide when medium was added to cells. Each
spectrum was obtained after ferricyanide had been in medium for 100 min.
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Fig. 7.
Effect of cells on ferricytochrome c
concentration in medium. Ferricytochrome
c (75 µM)-containing medium (Cyto
c) was added at time 0 to 2 cuvettes
either with or without DHAA (100 µM). Also shown is effect of
infusing AA (0.17 µmol · l 1 · min 1)
into medium containing ferricytochrome
c (75 µM) but no cells ( , a
positive control demonstrating direct reduction of ferricytochrome
c by AA). Two cell cuvettes contained
(mean ± SD) 89.9 ± 3.0 cm2
of endothelial surface, 1.42 ± 0.05 mg of protein, and 3.04 ± 0.07 LDH units/µg protein.
|
|
To determine whether DHAA also stimulated the extracellular reduction
of other electron acceptors by the endothelial cells, the effects of
DHAA on the endothelial cell uptake of methylene blue were determined
(Fig. 8). In contrast to ferricyanide,
which remains in the medium as ferrocyanide after reduction, when the methylene blue is reduced on the cell surface, the lipophilic reduced
form enters the cells, where it becomes sequestered or autooxidized
back to MB+ (14). Thus, when
methylene blue was used, only the oxidized form was found in the
medium. Because the first step in methylene blue uptake is reduction on
the endothelial surface (3, 15), the lack of effect of DHAA on uptake
suggested that different mechanisms are involved in the endothelial
reduction of ferricyanide and MB+.
However, in a cuvette experiment such as that depicted in Fig. 8, the
rate of methylene blue uptake is influenced by the rates of
intracellular sequestration and extracellular autooxidation as well as
by reduction (14).

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Fig. 8.
Effect of presence of DHAA or pretreatment with DHAA on endothelial
cell uptake of methylene blue. Medium containing methylene blue (10 µM) or methylene blue+DHAA (100 µM) was added at
time 0 to 2 cuvettes, but no DHAA was
added to third cuvette at time 0. To
third cuvette, DHAA (100 µM) was included in medium for 15 min before
time 0. Then cells were washed, and
methylene blue but no DHAA was added at time
0. Three cuvettes contained (mean ± SD) 76.9 ± 1.7 cm2 of endothelial surface,
2.95 ± 0.08 mg of protein, and 4.32 ± 0.17 LDH units/µg
protein.
|
|
To further evaluate the relative effects of DHAA on endothelial cell
ferricyanide and MB+ reduction,
the reduction of ferricyanide and
MB+ was studied in the cell
column. Under the transient reaction conditions in the column, the rate
of MB+ reduction is the
rate-limiting step in determining the
MB+ disappearance from the
perfusate (15). Again, DHAA stimulated ferricyanide reduction but had
little effect on MB+ reduction
(Figs. 9 and 10). Figure
9 shows column effluent concentration curves obtained after bolus
injections of ferricyanide before DHAA was added to the perfusate and
after the DHAA exposure. The amount of time the cells were exposed to
the ferricyanide was varied by varying the column flow rate. The
concentration curve for the reference indicator (FITC-dextran), which
is convected through the column without interacting with the cells,
reflects the concentration of ferricyanide that would have been
measured in each sample had there been no reduction of ferricyanide on passage through the column. For clarity of presentation, only one
reference indicator curve is shown because, except for small timing
differences due to the fact that each curve is from a separate bolus
injection, the reference curves were nearly superimposable with each
other and with the ferricyanide curves obtained before treatment with
DHAA. The bolus injections were carried out at three different flow
rates to maximize the possibility of observing any changes in reduction
rate that might occur under the assumption that if reduction rate were
rate limiting, the fraction reduced would be dependent on column
transit time. The time scale (x-axis) is normalized to the column mean transit time at each flow rate so that
the results from the different flows can be compared on the same
graph.

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Fig. 9.
Effect of DHAA on reduction of ferricyanide during passage through an
endothelial cell column. Results are shown for bolus injections carried
out at 3 flow rates before addition of DHAA to perfusate (control) and
after column had been perfused with perfusate containing 200 µM DHAA
for 30 min. Vertical axis is concentration of injected indicator
[either reference indicator FITC-dextran (Ref; solid line) or
ferricyanide (symbols)] divided by its injected amount (0.075 and
20 nmol for FITC-dextran and ferricyanide, respectively). Horizontal
axis is time after bolus injection divided by mean transit time through
column. This normalized time scale is used for this comparison because
time taken for bolus to pass through column is flow-rate dependent. For
clarity, only 1 of 3 nearly superimposable FITC-dextran curves is
shown. This cell column contained 154 cm2 of endothelial surface, 2.62 mg of protein, and 3.02 LDH units/µg protein.
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Fig. 10.
Effect of DHAA on reduction of methylene blue during passage through an
endothelial cell column. Results are shown for bolus injections at 3 flow rates before addition of DHAA to perfusate (control) and after
column had been perfused with perfusate containing 200 µM DHAA for 30 min. Vertical axis is concentration of injected indicator [either
FITC-dextran (Ref; solid line) or methylene blue (symbols)]
divided by its injected amount (0.075 and 2.8 nmol for FITC-dextran and
methylene blue, respectively). Horizontal axis is time after bolus
injection divided by mean transit time through column. For clarity,
only 1 of 3 nearly superimposable FITC-dextran curves is shown. This
cell column contained 156 cm2 of
endothelial surface, 2.31 mg of protein, and 3.28 LDH units/µg
protein.
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|
In the absence of DHAA, there was little separation between the
ferricyanide and reference indicator (FITC-dextran) curves, indicating
that little ferricyanide was reduced. After the DHAA exposure, the area
under the ferricyanide curve was considerably smaller than that under
the reference curve, indicating that the ferricyanide reduction was
much greater. The fraction reduced was in fact column transit-time
dependent, indicating that the reduction rate rather that the bolus
delivery rate was rate limiting. The results of the similar experiment
using MB+ as the electron acceptor
are shown in Fig. 10. In contrast to the ferricyanide, substantial
MB+ reduction occurred before the
addition of DHAA, and including DHAA in the perfusate had little
additional effect on MB+
reduction. To determine the time course and evaluate the persistence of
the DHAA effect in the cell column, several boluses were injected into
the cell column over a period of 105 min. The fractions of ferricyanide
and MB+ reduced on passage through
the cell column were determined before DHAA was added to the perfusate,
with DHAA in the perfusate, and after the DHAA had been washed out of
the perfusate (Fig. 11). The ferricyanide
reduction increased with time during the DHAA perfusion and remained
relatively stable after the DHAA had been washed out. The
MB+ reduction was relatively
unaffected by DHAA on the same time course.

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Fig. 11.
Fraction of injected ferricyanide or methylene blue reduced during
passage through endothelial cell column vs. time. DHAA (200 µM) was
included in perfusate between 16 and 72 min (i.e., between vertical
dashed lines). Ferricyanide cell column contained 118 cm2 of endothelial surface, 1.94 mg of protein, and 3.28 LDH units/µg protein. Methylene blue cell
column contained 136 cm2 of
endothelial surface area, 1.93 mg of protein, and 3.12 LDH units/µg
protein.
|
|
 |
DISCUSSION |
These results are consistent with the hypothesis that there is a
reductase on the bovine pulmonary arterial endothelial cell surface
that can reduce extracellular ferricyanide and utilize intracellular AA
as a direct electron donor or perhaps indirectly as a component of an
intracellular electron transport sequence. According to this
hypothesis, the explanation for the stimulatory effect of DHAA is that
DHAA is transported into the cells where it is reduced to AA. Thus the
intracellular reduction of DHAA provides AA for the reduction of the
ferricyanide via a transplasma membrane electron transport system.
Recycling of DHAA to AA within the cells would account for both the
persistence of the DHAA effect and the ability of the cells to reduce
more ferricyanide than predicted from the stoichiometry of DHAA uptake
alone. The lack of effect of DHAA on the previously described (2, 3,
16) thiazine reductase activity suggests two separate electron
transport systems on the endothelial cell plasma membrane, each with
different electron donor and acceptor preferences.
Ferricyanide reduction via transplasma membrane electron transport has
been observed in several mammalian cell types (8, 10, 15, 26), and a
DHAA-stimulated ferricyanide reduction system has been observed in
erythrocytes (17). Orringer and Roer (17) concluded that the DHAA
stimulation of the reduction of ferricyanide in the presence of
erythrocytes was the result of intracellular DHAA reduction to AA,
which then left the cells, resulting in nonenzymatic reduction of the
ferricyanide in the medium. Inside cells of various types that have
been studied (18, 19), including endothelial cells (9), DHAA is, in
fact, rapidly reduced to AA. That explanation would also be consistent
with the fact that ferricyanide is rapidly reduced directly by AA. McGown et al. (13) came to a similar conclusion with respect to the
ability of DHAA to stimulate extracellular methemoglobin reduction by
erythrocytes. On the other hand, May et al. (12) carried out studies on
erythrocytes using nitro blue tetrazolium, which is not directly
reduced by AA, as the electron acceptor. They concluded that the nitro
blue tetrazolium reduction by AA-loaded erythrocyte ghosts was due to
the ability of intracellular AA to donate electrons to a transplasma
membrane oxidoreductase. Schipfer et al. (20) also demonstrated that
DHAA stimulation of a ferricyanide reductase in erythrocytes could
occur without release of a reducing agent into the medium.
There are several lines of evidence suggesting that DHAA stimulation of
the endothelial ferricyanide reductase in the present study did not
involve direct access of AA produced in the cells to the external
ferricyanide. For example, the total amount of DHAA taken up by the
endothelial cells was much less than the amount of ferricyanide
reduced. The rate of DHAA uptake in Fig. 4 was only ~3% of the rate
of ferricyanide reduction stimulated by the same concentration of DHAA
in Fig. 3. Thus, with the assumption that AA is the electron donor, it
would have to be recycled at a rapid rate to account for the observed
ferricyanide reduction. The conclusion from the relatively slow rate of
DHAA uptake is that the recycling must be intracellular, since the DHAA
uptake rate would not be sufficient to support the rate of ferricyanide reduction if it involved oxidation of AA that had been released from
the cells. This conclusion is further supported by the observation that
DHAA-containing medium conditioned by exposure to the cells did not
reduce ferricyanide. An AA concentration sufficient to make a
significant contribution to the ferricyanide reduction would have been
clearly detectable by UV absorption, but conditioning of the medium by
the DHAA-treated cells did not increase the UV absorbance of the
medium.
To evaluate the possibility that AA released from the cells might be
too short-lived to be detectable in cell-conditioned medium removed
from the cells, we took two approaches. One was to add AO to the medium
to scavenge any AA that might be released into the medium bathing the
DHAA-stimulated cells. The lack of effect of AO on DHAA-stimulated
ferricyanide reduction suggests that AA in the medium was not involved.
The other approach was to examine the ability of DHAA-stimulated cells
to reduce ferricytochrome c. Because
ferricytochrome c was directly reduced
by AA- but not by DHAA-stimulated cells, a role for extracellular AA
again seems unlikely.
The greater stimulatory effect of DHAA compared with AA is apparently
due to the faster rate of DHAA accumulation by the cells (Fig. 4).
Endothelial cells have been found to transport AA and DHAA (9, 23). In
various cell types, AA and DHAA have been found to be transported by
different mechanisms (5), and DHAA uptake has generally been faster
than AA uptake (5). Wilson et al. (23) could not detect de
novo synthesis of AA or a basal level of AA in cultured rat muscle
microvascular endothelial cells, suggesting that endothelial cells, or
at least endothelial cells in culture, do not synthesize AA, even when
they are from species with hepatic AA synthesis. AA is not generally
considered to be a necessary constituent of cell culture media (5), and
the AA concentration in the endothelial cell growth medium used in the
present study is negligible (11). The H-H buffer solution used in the
study had no AA except when AA was specifically added. If there were
any AA in the cells before the addition of DHAA or AA to the H-H
solution, it was apparently either not sufficient or not available to
allow the ferricyanide reductase to operate at a significant fraction
of its capacity.
The effect of DHAA was apparently saturable (Fig. 4). Under the
assumption that the stimulatory effect of DHAA involves its uptake into
the cells followed by intracellular reduction to AA, this suggests
that, for a given extracellular ferricyanide concentration, once the
intracellular AA has reached a certain level within the cells, its role
is permissive rather than rate limiting. This result is similar to that
for DHAA stimulation of erythrocyte ferricyanide reduction (17).
Interestingly, the concentration of DHAA in the medium that resulted in
a half-maximal reduction rate by these endothelial cells of ~0.1 mM
is on the same order as the value of ~0.04 mM that can be estimated
for erythrocytes from the data of Orringer and Roer (17).
The data are consistent with the hypothesis that DHAA is reduced
intracellularly to AA, which is an electron donor for the ferricyanide
reductase. They are also consistent with the observation that
erythrocytes can utilize intracellular AA to reduce extracellular ferricyanide (12). However, it is conceivable that DHAA stimulates the
ferricyanide reductase through some other pathway. Schipfer et al. (20)
noted stimulation of a ferricyanide reductase in the erythrocyte plasma
membrane when DHAA was present in the medium. They concluded that NADH
was the intracellular electron donor and provided no explanation for
the DHAA effect.
In the cuvette experiments, the uptake of methylene blue by the
endothelial cells is determined by the rates of the intracellular sequestration reaction and extracellular autooxidation in addition to
the rate of reduction on the cell surface (14). On the time course of
the transient passage of the bolus through the cell column, the surface
reduction determines the rate of disappearance of methylene blue from
the perfusate (16). The cell column results confirmed the selectivity
of the DHAA-stimulated reductase activity with respect to the two
electron acceptor probes ferricyanide and methylene blue, revealing
that there are two distinct systems involved in the reduction of these
two acceptors. One is a thiazine reductase that does not require
intracellular AA, and another is a ferricyanide reductase that requires
intracellular AA. Neither requires the release of AA into the medium.
The ability to distinguish between the two systems does not necessarily
mean that they are unrelated. Electron transport chains typically have
multiple prosthetic groups, such that different electron donors and
acceptors participate at different sites along the chain. Mitochondrial
electron transport is prototypical in this regard. It is not clear how
complex endothelial transplasma membrane electron transport systems
might be. Villalba et al. (21) have begun to dissect the monoDHAA
reductase of the hepatocyte plasma membrane, which appears to involve a
cytochrome b5
reductase on the intracellular plasma membrane surface, a coenzyme Q
membrane component, and an as yet unidentified component(s) at the
outer surface. Thus there is precedent for expecting a multicomponent
electron transport chain within the endothelial membrane. On the other
hand, several different transplasma membrane electron transport systems
have been characterized in other mammalian cells (7), and there is
evidence for an iodonium-sensitive NAD(P)H oxidase-like enzyme in the
endothelial plasma membrane (25) in addition to the ferricyanide and
thiazine reductases. Thus completely separate systems are also
possible.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-24349, by a grant from the American Heart
Association, and by the Department of Veterans Affairs.
 |
FOOTNOTES |
Address for reprint requests: C. A. Dawson, Research Service 151, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI
53295-1000.
Received 21 November 1997; accepted in final form 21 January 1998.
 |
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