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Oxidants, ATP Depletion, and Endothelial Permeability to Macromolecules
By Jeff Wilson, Michael Winter, and D. Michael Shasby
Oxidants can reversibly increase the permeability of endothelium to ions and macromolecules.Oxidants also deplete
ATP in cultured endothelial cells. We asked if oxidantmediated ATP depletion, alone, accounted for the effects
of oxidants on endothelial permeabilityto macromolecules.
When porcine pulmonary artery endothelial cells were
exposedto 2.5 mmol/L H,O,, ATP was depleted to 31.7% f
1.8% of control within 15 minutes and was reduced to
23.1 % k 2.0% of control after 30 minutes. To determine if
this magnitude of ATP depletion could account for the
oxidant-induced increase in endothelial permeability to
macromolecules, we measured ATP in endothelial cells
exposed to metabolic inhibitors of ATP production. W e
then measured the effects of these metabolic inhibitors on
endothelial monolayer permeability to macromolecules.
ATP levels were reduced to 44% & 4% of control by 12
mmol/L deoxyglucose (DOG) in the absence of glucose and
to 2% 2 1.3% of control by DOG with 25 nmol/L antimycin
A in the absence of glucose. Reduction of endothelial cell
ATP to these levels with the metabolic inhibitors did not
alter the flux of albumin or dextran across the endothelial
monolayers.Thus ATP depletion, by itself, does not explain
oxidant-induced changes in endothelial permeability to
macromolecules.
0 1990 by The American Society of Hematology.
I
reversible oxidant-induced increases in endothelial permeability to albumin.' We then caused similar levels of ATP
depletion in the same cells with deoxyglucose and antimycin
A and asked if these levels of ATP depletion altered the flux
of albumin or dextran across monolayers of the same cells.
NCREASED PERMEABILITY of the microvasculature
is an important characteristic of acute inflammatory
edema.' Granulocytes are believed to play an important role
in this process, and reactive oxygen molecules (oxidants)
released from granulocytes have been shown to increase
endothelial permeability in vitro and in vivo.2s3
ATP depletion occurs in endothelial and other cells exposed to oxidant^.^ The precise mechanisms responsible for
ATP depletion have not been determined, but several pathways are probably involved.5 The potential significance of
ATP depletion in oxidant injured endothelial cells to the
pathogenesis of acute inflammatory edema is also not known.
Holmsen found that H,O, depleted ATP in platelets, but that
ATP depletion to the level caused by H,O, did not disrupt
platelet function if the adenylate energy charge were not
altered.6 Wysolmerski reported that ATP depletion with
deoxyglucose and antimycin A caused subtle changes in the
staining pattern of the peripheral band of actin in cultured
endothelial cells. The same ATP depletion prevented the
cells from contracting after exposure to histamine, cytochalasin B or ethchlorvynol.' Bolin et a1 blocked glycolytic ATP
production in perfused rabbit lungs and found that inhibition
of glycolysis did not alter the hydraulic conductivity of the
lung vasculature.8
From such scant information it is not possible to predict
what the effects of oxidant-mediated ATP depletion might be
on endothelial permeability to macromolecules. In these
experiments we measured the extent of oxidant-induced ATP
depletion in porcine pulmonary artery endothelial cells,
cultured the same as the cells we previously used to observe
~~
From the Department of Internal Medicine. University of Iowa
College of Medicine, Iowa City, IA.
Submitted January 18. 1990; accepted August 24, 1990.
Supported by NIH Grants No. HL33540. HL 36605, and
HL14230.
Address reprint requests to Jeff Wilson, MD. Department of
Internal Medicine, University of Iowa College of Medicine, Iowa
City, IA 52242.
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7612-0023$3.00/0
2578
MATERIALS AND METHODS
Materials. Materials were obtained form the following sources:
H,O,, trichloroacetic acid (TCA), and phosphoric acid from Fischer
Scientific, Pittsburgh, PA; antimycin A (AA), dextran (average
molecular weight 67,900), glucose, deoxyglucose (DOG), bovine
serum albumin (BSA, fatty acid free), luciferin-lucerferase, and
fetal bovine serum (FBS) from Sigma Chemical, St Louis, M O
catalase was also from Sigma and was pretreated with polymyxin B
agarose prior to use; I4C albumin from New England Nuclear,
Boston, MA; 'H dextran from Amersham, Arlington Heights, IL;
glucose free Earle's balanced salt solution (EBSS-GF) was made in
the laboratory using the standard ion constituents for Earle's
balanced salts; Medium 199 (M199) and trypsin-EDTA were from
the Cancer Center, University of Iowa.
Cell culture. Porcine pulmonary arteries were obtained immediately after slaughter at a local abattoir. Resected ends of the arteries
were clamped, and the artery segment was quickly dipped in 70%
ethanol, then immediately rinsed in M199 with penicillin (100
U/mL) and streptomycin (100 pg/mL) (1 x P + S). Arteries were
then undamped and placed in sterile M199 with 1 x P + S. In the
laboratory the arteries were opened longitudinally and the lumen
gently scraped with a scalpel blade or a cotton tipped applicator.
Cells were released into 35-mm-diameter tissue culture plates with
M199 supplemented with Basal Medium Eagle (BME) vitamins
(GIBCO, Grand Island, NY) and amino acids, 1 x P + S, 10% FBS
and then incubated at 37OC in 5% CO, and 95% air. Cells were
subcultured 3: 1 using trypsin-EDTA twice weekly, and cultures
from passages 4 through 10 were used. Cells were determined to be
endothelial by morphology and by uptake of acetylated low density
lipoprotein."
ATP measurements. Porcine pulmonary artery endothelial cell
monolayers were exposed to H,O, in M199 or to combinations of
metabolic inhibitors with and without glucose in EBSS. Monolayers
were then rinsed with saline or EBSS-GF at 4OC and extracted with
4OC 6% trichloroacetic acid (TCA). After refrigeration at 4OC for 1
hour, ATP was measured by either a luciferin-luciferase lightproducing assay or by HPLC. For the luciferin-luciferase assay each
plate was scraped with a rubber policeman, transferred to a 1.5 mL
microcentrifuge tube and centrifuged at 1,750g for 5 minutes.
Neutralizing the extracts before assaying did not change the
measured ATP values and was not done. The buffered firefly lantern
extract was incubated overnight in distilled water at 4OC. Before use
Blood, Vol 76, No 12 (December 15). 1990: pp 2578-2582
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ATP DEPLETION AND ENDOTHELIAL PERMEABILITY
the extract was centrifuged at 1,900g for 10 minutes and the
decanted supernatant mixed 2:1 with a buffer of sodium arsenate
(100 mmol/L) and magnesium chloride (40 mmol/L) to a total
volume of 25 mL. A standard stock solution of ATP (10 mmol/L)
mixed in 10% TCA was diluted to yield standards of 5 to 25 pmol/L
ATP. The reaction mixture consisted of 2 mL enzyme/buffer mixed
with 50 pL sample or ATP standard at room temperature. Luminescence was measured 30 seconds after mixing from a continuous
recorder linked to an Aminco Fluoro-Colorimeter (American Instrument Company, Silver Spring, MD)?
The HPLC assay of ATP used the method of Tekkanant et al."
The adenine nucleotides were separated on an ion-pairing reversed
phase C18 column using a mobile phase of 60 mmol/L KH2P0,,
0.45 mmol/L tetrabutylammonium phosphate, and 3.6% acetonitrile (pH 3.2). Samples were precipitated with 6% TCA at 4"C,
neutralized with 1 volume 20% tri-n-octylamine in Freon 113; the
aqueous phase was then filtered through a 0.45 pmol/L nitrocellulose filter. The pH was adjusted to 3.2 with phosphoric acid, 1.5 mL
samples were injected onto the column and eluted at a flow rate of 1
mL/minute using a Beckman high performance liquid chromatography system (Beckman Instruments, San Ramon, CA). Peaks were
detected at 254 nm and quantitated by comparison with peak heights
of standards run on the same day.
Albuminflux measurements. Porcine pulmonary artery endothelial cells were plated onto 6.5-mm-diameter Transwell polycarbonate membranes (precoated with 30 pg/mL fibronectin for 30
minutes) with 0.4 pm pores (Costar, Cambridge, MA). Cells were
cultured in MI99 with 10% FBS and used on day 3 after plating.
During the control period each monolayer was rinsed twice with
EBSS and then 150 pL of EBSS with 145 pmol/L albumin (cold);
-33,000 cpm/mL of I4Calbumin was added to the luminal chamber.
Each monolayer was then placed into the well of a 24-well tissue
culture plate containing 700 pL EBSS (these volumes balanced the
fluid levels in the chambers), and incubated for 40 minutes at 37OC
in 5% CO,, 95% air. Each monolayer was then removed from the
plate and 500 pL of the medium taken from the well and counted in
the scintillation counter. For the second 40-minute experimental
period, each monolayer was rinsed three times in EBSS-GF. One
hundred fifty microliters of EBSS with or without 5.5 mmol/L
glucose, 25 nmol/L antimycin A and/or 12 mmol/L deoxyglucose
with 145 pmol/L albumin and 33,000 cpm/mL I4C albumin was
then added to the luminal compartment of each monolayer that was
placed into the well of a 24-well tissue culture plate containing 700
pL of the same solution without the albumin. After 40 minutes the
monolayer was removed and the lower chamber sampled as in the
control period. Albumin flux was calculated as follows:
counts through the monolayer
= albumin flux in pmoles/min/
cpm/pmole X 40 min X 0.331 cm2
cm'.
Dextranflux measurements. These data were determined in the
same manner as were the albumin flux measurements except that 1
mg/mL dextran (average molecular weight 67,900, 14.7 pmol/L)
and 145 pmol/L albumin were added to both sides of the monolayers, and 'H dextran (average molecular weight 67,900, approximately 235,000 cpm/mL) was added to the luminal side. In
addition, the monolayers were incubated with the metabolic inhibitors for 15 minutes before initiating the measurement of the second
dextran flux. For the experiments in which monolayers were exposed
to H,O,, the dextran flux over a 40-minute control period was
measured, the monolayers were exposed to the indicated H,O,
concentration for 15 minutes, then treated with 10,000 units
catalase, washed, and a second 40-minute flux of dextran was
measured.
2579
Statistical analysis. All data are presented as the mean *
standard error unless otherwise indicated. Changes in albumin or
dextran flux were analyzed by analysis of variance, and differences
between groups were tested using a Tukey HSD post hoc test of
pairwise mean differences. Differences were considered significant at
the P < .05 level.
RESULTS
Effects of H202
on ATP levels in endothelial cells. We
previously reported that oxidants reversibly increased albumin flux across porcine pulmonary artery endothelial cell
monolayers after a 15-minute expos~re.'.'~When similar
endothelial cells were exposed to H202 concentrations between 100 rmol/L and 2.5 mmol/L, there was a significant
decrease in ATP after 15 minutes to approximately 25% of
control values (Fig 1). When catalase was added after a
15-minute exposure to 2.5 mmol/L H202,ATP levels were
23.1% f 2.0% of control after 30 minutes (Fig 2). The
threshold concentration of H20, necessary to cause a decrease in ATP was between 10 and 50 pmol/L (Fig l). These
dose-response relationships are similar to those reported by
others: The degree of ATP depletion was similar between 50
pmol/L and 2.5 mmol/L H202after 15 minutes.
Eflects of metabolic inhibitors on ATP levels. If oxidantinduced increases in macromolecule flux across endothelial
monolayers are caused by oxidant-induced ATP depletion,
then quantitatively similar depletion of ATP with metabolic
inhibitors would be expected to alter macromolecule flux
across the endothelial monolayers. To assess this, we first
measured the extent of ATP depletion caused by some
inhibitors of ATP production. Incubating endothelial cells in
glucose-free media caused a gradual, small decline in ATP
levels. At 20 minutes ATP levels were 92% 1.2% of control
and 77% f 6.6% of control at 40 minutes in endothelial cells
incubated in glucose-free EBSS. Deoxyglucose (12 mmol/
L), an inhibitor of glycolytic ATP production, caused a
significant decrease in ATP in the absence of glucose at 10,
20 and 40 minutes (Fig 3). When deoxyglucose was com_+
120
f'
e
100
0
80
1
c
c
k 0
aE
60
Q)
0
t
40
Q
W
20
0
.01 .05 .10 .25 .50 1.0 2.0 10.0
H202 (mM)
Fig 1. ATP levels in porcine pulmonary artery endothelial cells
exposed to graded concentrations of H,O, for 15 minutes. Results
are expressed as % of control value = 34.9 f 2.5 pmoles ATP/pg
cell protein (n 2 3 for each value).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2580
WILSON, WINTER, AND SHASBY
120
a
z
I-
a za,
20
80
60
10
0
L
a,
40
Q
U
20
0
0 Min
15 Min
30 Min
0
60 Min
con
DOG
AA
DOG
Fig 2. ATP levels in porcine pulmonary artery endothelial cells
exposed t o 2.5 mmol/L H202.Catalase (10,OOO u n i d t b m m diameter well) was added at 15 minutes. Results are expressed as
% of control value = 34.9f 2.5 pmoles ATP/pg cell protein (n z 3
for each value).
bined with antimycin A, an inhibitor of mitochondrial ATP
production, in the absence of glucose, ATP was depleted to
7% 3% of control within 10 minutes, and to 2% 1.3% of
control within 20 minutes (Fig 3).
Efects of metabolic inhibitors on macromolecule flux.
We next evaluated the effects of metabolic inhibitors of ATP
depletion on the flux of macromolecules across endothelial
monolayers. None of the combinations of metabolic inhibitors caused a significant change in albumin flux across the
endothelial monolayers (Fig 4). In particular the combination of antimycin A and deoxyglucose, which led to the most
extensive ATP depletion, caused no change in the albumin
flux.
Because there is controversy' about how albumin crosses
endothelium, we also measured the effect of the metabolic
inhibitors on the flux of dextran across endothelial monolayers. Deoxyglucose caused a small but statistically significant
-
h
-
'O1
T
EA
Treated
E
52
c
L .-
2
=.E aE
C
I-
Con
e4
c
n
decrease in the dextran flux. The change in the flux of
dextran across monolayers treated with antimycin A and
deoxyglucose was not different from the change in the flux
across control monolayers (Fig 5).
In contrast to the lack of effect of metabolic inhibitorinduced reductions in ATP on transendothelial macromolecule flux, treatment of endothelial monolayers with 2.5 and
5.0 mmol/L H202 for 15 minutes increased the flux of
dextran across the monolayers (Fig 6). ATP in monolayers
from the same passage of cells and exposed to the same
concentrations of H,O,for 15 minutes were 6 f 0% and 5 f
0.6% of control at 15 minutes and 28 f 0.2% and 25 1.1%
n
Con
-DOG
l2O1
Albumin flux across porcine pulmonary artery endotha
lial cell monolayers during a Gminutecontrol period and then 40
minutes after exposure t o EBSS-GF (Con), DOG (12 mmol/L) in
EBSS-GF or DOG (12 mmol/L) and antimycin A (AA, 25 nmol/L) in
EBSS-GF. There were no significant differences between the
responses of the control and the treatment monlayers (n = 6 for
each group).
0
0
-a,
ac
C
x-
a,
0
Q
L.
v
a,
Q
v
204
0
1
0
t
,
....
+-.P--:"':"."":
I......
20
40
:
.
.
.
:
.
.
e
60
80
,
,
,
100
Time (minutes)
Fig 3. ATP levels in porcine pulmonary artery endothelial cells
exposed t o EBSSGF (Con). deoxyglucose (12 mmol/L. DOG) in
EBSS-GF. or DOG (12 mmol/L) and antimycin A (25 nmol/L, AA) in
EBSS-GF for indicated times. Results are expressed as % of
control = 26.7 i 2.5 pmoles ATPIpg cell protein (n 2 3 for each
value).
con
DOG
AA
DOG
Fig 5. Dextran flux across porcine pulmonary artery endotha
lial cell monolayers during a 40-minute control period and then 40
minutes after a 15-minute preexposure t o EBSS (Con). deoxyglucose (12 mmol/L. DOG) in EBSS-GF or DOG (12 mmol/L) and
antimycin A (AA, 25 nmol/L) in EBSSGF. The DOG group had a
small but statistically significant decrease in dextran flux compared with the controls. The response of the AA-DOG group was
not different from that of the controls (n 2 6 for each group).
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2581
ATP DEPLETION AND ENDOTHELIAL PERMEABILITY
Con
12
Treated
T
10
8
6
4
2
0
Con
2.5 H202
5.0 H202
Dextran flux across porcine pulmonary artery endothe
lial cell monolayers during a 40-minute control period and then 40
minutes after a 16minute exposure to Medium 199 or Medium
199 with 2.5 or 6.0 mmol/L H,O,. After the 1Sminute exposure all
monolayers were exposed to catalase (l0,OOO units), washed and
the second 40-minute flux was measured. The increase in flux
across monolayers exposed to 2.5 and 5.0 mmol/L H,O, was
greater than that across controls. The increase in flux across the
monolayers exposed to 5.0 mmol/L H,O, was greater than the
increase in flux across monolayers exposed to 2.5 mmol/L HZ02.
of control at 60 minutes, for 2.5 and 5.0 mmol/L H20,
respectively.
DISCUSSION
Oxidants cause ATP depletion in cultured endothelial
cells: They increase the permeability of cultured endothelial
monolayers to macromolecules and the permeability of an
intact microvasculature to ions.399However, the relationship
between ATP depletion and the changes in permeability have
previously not been directly addressed. Hinshaw et a1 found
that ATP depletion, via glucose deprivation, to 10% of
control levels over 3 hours resulted in progressive shortening
and aggregation of microfilaments accompanied by retraction and rounding in P388 murine lymphoma cells.12 On the
other hand, Wysolmerski found that a similar degree of ATP
depletion in endothelial cells treated with antimycin A and
deoxyglucose caused only subtle and infrequent changes in
cell-to-cell contacts and prevented cell retraction in response
to histamine, cytochalasin B and ethchlor~ynol.~
Holmsen et
a1 observed that platelets depleted of ATP to approximately
25%of control with H202retained a normal shape-change
response when exposed to ADP.6 More recently Hinshaw
showed that endothelial cells depleted of ATP to less than 5%
of control levels with metabolic inhibitors underwent a shape
change only after subsequent exposure to the ionophore
A23 1 87.14However, ATP depletion by itself did not change
endothelial cell shape; similar changes in endothelial cell
shape after exposing endothelial cells to A23187 have been
reported with A23187 alone, without ATP depletion? Thus
the role of ATP in the cytoskeletal control of cell shape is
complex and may vary among cell types.
In this investigation we found that depletion of ATP with
metabolic inhibitors, to 4%
of control levels, did not
increase albumin or dextran flux across endothelial cell
monolayers over 40 minutes. This is a time course similar to
that used in our current and prior experiments when we
observed that oxidants reversibly increased albumin flux
across similar monolayers?*” It is also similar to the time
course for the reversible increase in ionic conductance of
cerebral microvessels exposed to oxidants, as reported by
O l e ~ e nIt
. ~is possible that if such severe ATP depletion had
been extended for several hours, the macromolecule flux
might have changed, although it is not clear that this would
have represented a reversible effect.
The degree of ATP depletion seen with antimycin A and
deoxyglucose in the absence of glucose ( 4 %of control) was
as great or greater than that seen with oxidant doses capable
of increasing the flux of albumin across the endothelium.
Xanthine and xanthine oxidase (10 mU/mL) increased the
flux of albumin across endothelial monolayers after a 15minute exposure? ATP levels in endothelial monolayers
exposed to 10 mU/mL xanthine oxidase have been reported
to be greater than 50% of control after 30 minutes and 1
hour.” Similarly, 10 mU/mL glucose oxidase increased
endothelial monolayer permeability to albumin. In the same
study, the same doses of glucose oxidase decreased endothelial ATP to approximately 75% of control levels.16 Exposing
endothelial monolayers to 2.5 mmol/L H202for 15 minutes
increased both transendothelial albumin flux in our prior
study and transendothelial dextran flux in the current
in~estigation.’~
However, endothelial cell ATP was greater
after 2.5 mmol/L H202than after antimycin A and deoxyglucose. Because antimycin A and deoxyglucose treatment did
not increase transendothelial macromolecule flux, oxidantinduced ATP depletion alone does not explain the increase in
transendothelial macromolecule transfer seen after endothelial exposure to oxidants.
In related experiments Bolin et a1 found that inhibition of
glycolysis in an isolated perfused rabbit lung model did not
alter the hydraulic conductivity of the lung? Oxidative ATP
production was not blocked, nor were ATP levels measured
in Bolin’s study, so the level of ATP depletion achieved is not
known. However, Bolin’s observations are consistent with our
own finding that inhibition of glycolysis with deoxyglucose
did not increase macromolecule flux across the endothelium.
Rasio et a1 found that the combination of iodoacetic acid
(IAA), potassium cyanide and hypoxia increased capillary
permeability in the eel s~imb1adder.I~
However, these agents
have effects independent of ATP depletion that may contribute to changes in capillary permeability. IAA inhibits glycolysis by acetylating sulfhydryl groups in glyceraldehyde
phosphate dehydrogenase. However, IAA reacts with any
susceptible sulfhydryl groups and has been shown to cause
increases in vascular permeability independent of its effects
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WILSON, WINTER, AND SHASBY
2582
on ATP.' In addition, hypoxia has been demonstrated to
increase capillary permeability by the generation of free
radicals.''
Although ATP depletion alone does not increase macromolecule flux across endothelial monolayers, ATP depletion may
contribute to the oxidant-induced changes in permeability.
Oxidants cause phospholipase C hydrolysis of phosphatidylinositol in endothelial cells.I3 ATP depletion has been demonstrated to potentiate the phospholipolysis that occurs after
exposing cells to phospholipase C, and oxidant ATP depletion may potentiate oxidant-stimulated phospholipase C
activity."
In summary, we do not find that ATP depletion alone can
explain oxidant-induced changes in the endothelial barrier to
macromolecules. However, oxidants cause simultaneous
changes in many cell functions. ATP depletion may act in
concert with other pathways to alter endothelial barrier
function.
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1990 76: 2578-2582
Oxidants, ATP depletion, and endothelial permeability to
macromolecules
J Wilson, M Winter and DM Shasby
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