 |
INTRODUCTION |
Free radicals and their secondary products have been implicated in
many types of vascular disorders, including ischemia-reperfusion injury, myocardial infarction, hypertension, and atherosclerosis (1-3). Xanthine oxidoreductase
(XOR,1 EC 1.2.3.2) is a key
source of reactive oxygen species in the intravascular compartment,
with xanthine oxidase primarily viewed to be an intracellular source of
reactive species and (XO) inhibition showing protective effects toward
both in vitro and in vivo model systems.
Importantly, salutary actions of XO inhibition also occur at sites
remote from the original source of injury. Allopurinol, or pretreatment
of animals with the molybdopterin enzyme inhibitor tungsten to
inactivate XO, decreases injury to lung vascular barrier function
secondary to splanchnic ischemia-reperfusion (4, 5). Also, allopurinol,
which exhibits no direct antioxidant properties at pharmacologic
concentrations, displays tissue-protective actions in organ systems low
in or devoid of detectable endogenous XOR activity (6). For example,
both rabbit and clinical myocardial ischemia-reperfusion injury was
significantly attenuated by allopurinol, despite several reports
revealing that rabbit and human heart have low to undetectable amounts
of XO (7-10). It therefore becomes critical to understand the tissue
distribution of XO and underlying mechanisms of cell injury that are
mediated by a source of reactive species that is widely implicated in
various pathological processes.
The ability of circulating XO to bind to vascular cells of remote
organs may explain the efficacy of XO inhibitors in protecting organs
having low XOR activity. Plasma levels of XO rise following ischemia-reperfusion injury to the splanchnic system or following hypovolemic shock, with further increases in plasma XO observed upon
perfusion with high concentrations (1000 units/h) of intravenous heparin (4, 11, 12). Lung-associated XO activity increases following
perfusion with XO-rich effluent from reperfused ischemic liver (13).
Thus, during diverse pathologic processes, XO released into plasma from
tissues replete in XO specific activity (e.g. the splanchnic
system) can circulate to remote sites and bind to target tissues low in
or devoid of XO activity. Cell-bound XO may then be concentrated
several thousand-fold at the cell surface or interstitial matrix, where
its oxidant products could more readily react with cellular target
molecules and disrupt vascular functional and barrier properties. High
concentrations of XO associate with endothelium and bind to
heparin-Sepharose 6B complexes in vitro, but this phenomenon
has not been well characterized (11, 14, 15). Herein, we show that
endothelial-bound XO retains similar catalytic properties as XO in
solution, is initially associated with sulfated cell surface
proteoglycans, migrates to intracellular compartments via endocytosis,
generates reactive species not readily accessible to CuZn-SOD, and
finally, impairs vascular cell signal transduction.
 |
EXPERIMENTAL PROCEDURES |
Materials
Sephadex G-25 PD-10 columns were from
Amersham Pharmacia Biotech. Fetal bovine serum and defined
iron-supplemented calf serum were from Hyclone Laboratories, Inc.
(Logan, UT). All other cell culture media, Hanks' balanced salt
solution (HBSS), and trypsin were from Life Technologies, Inc. Xanthine
oxidase (bovine cream) and CHAPS were obtained from Calbiochem.
CuZn-SOD was from Oxis, Inc. (Portland, OR). Recombinant human EC-SOD
was prepared as described previously (16). Polyethylene
glycol-derivatized CuZn-SOD was from Sterling Drug, Inc. (Malvern, PA).
cGMP EIA kits were from Cayman Chemicals (Ann Arbor, MI).
Glycosaminoglycan lyases were purchased from Seikagaku Kogyo Co., Ltd.
(Tokyo, Japan). Heparin (porcine intestinal mucosa) was obtained from
Polysciences, Inc. (Warrington, PA). All other chemicals were from
Sigma. The carboxyl terminus of the human XO sequence (17) was selected for antigen production to ensure that the resultant polyclonal rabbit
antiserum would recognize xanthine oxidase generated by proteolytic
cleavage of the amino terminus of XDH. Polyclonal antibodies against
this recombinant human XOR fragment were developed (18). The carboxyl
terminus of human XOR was amplified by polymerase chain reaction
corresponding to the 358 carboxyl-terminal codons common to both XDH
and XO. The XOR polymerase chain reaction product was cloned into an
expression vector, induced for expression of the XOR 42-kDa fusion
product. The fusion protein was purified to near homogeneity and
injected into rabbits for production of polyclonal antibodies. A unique
specific signal corresponding to the recombinant XOR fusion product was
observed by Western analysis for all dilutions of anti-XOR sera.
Preimmune serum was not immunoreactive for XOR. This antiserum does not
cross-react with human, rat, bovine, or rabbit IgG or lactoferrin,
previously noted to be a problem for other antisera to XOR (19).
Cell Culture--
Bovine aortic and human umbilical vein
endothelial cells were isolated as described previously (20). Primary
cell culture preparations, subcultures, and cell removal from flasks
were conducted in the absence of proteases using scraping techniques.
Collagenase (0.1% in HBSS) was used only for preparing primary cell
isolates from human umbilical veins. Cells were propagated by
subculturing in a 1:4 ratio in medium 199 containing 5% fetal calf
serum, 5% iron-supplemented and defined calf serum, and 10 µM thymidine in 75-cm2 flasks. Cells
exhibited typical endothelial cobblestone morphology by phase contrast
microscopy and positive immunostaining for factor VIII antigen. For
XO-cell binding studies, cells between passages 5 and 8 were seeded at
a 1:4 split ratio in 12-well plates (4 cm2) or
25-cm2 flasks and used within 30 h of reaching
confluency. Primary cultures of fetal rat lung fibroblasts and type 2 alveolar epithelial cells were prepared as before (21).
Xanthine Oxidase Preparation and Assay--
Xanthine oxidase was
further purified by Sephadex G-25 chromatography to remove ammonium
sulfate and possible contaminating proteases. Xanthine oxidase was
monitored for protease contamination and, prior to its addition to cell
systems, activity was determined spectrophotometrically by the rate of
uric acid formation in 0.1 mM xanthine, 50 mM
potassium phosphate, pH 7.4, at 295 nm (
= 1.1 × 104 M
1·cm
1),
revealing a specific activity of typically ~1.4 units/mg of protein.
For some experiments, XO was inactivated by 10 mM KCN for
15 min. The enzyme was then dialyzed extensively against 50 mM potassium phosphate, pH 7.4, and assayed for residual
activity. Xanthine oxidase was labeled with 125I using
IODO-GEN from Pierce. Briefly, 1 mg of XO in 1 ml of 50 mM
potassium phosphate buffer, pH 7.4, 5 mCi of [125I]NaI,
and 0.3 µM KI were incubated with 0.1 mg of IODO-GEN for 10 min at 4 °C. The free 125I was separated from the
125I-XO by size exclusion chromatography using a Sephadex
G-25 PD10 column equilibrated with 50 mM potassium
phosphate, pH 7.4, followed by dialysis at 4 °C with four changes of
1 liter of 50 mM potassium phosphate, 25 µM
potassium iodide, pH 7.4, and dialysis with four changes of 1 liter of
50 mM potassium phosphate, pH 7.4. Greater than 95% of the
counts were precipitable with 10% trichloroacetic acid. For some
enzymatic analyses of XO-dependent reduction of oxygen,
succinoylated cytochrome c was synthesized as previously described (22).
Xanthine Oxidase-Cell Binding Studies--
To assay cell-bound
XO activity, cells in 75-cm2 flasks were rinsed twice with
HBSS and incubated at 37 °C with 15 ml of 0-10 milliunits/ml XO for
3 h in M199(
), defined as medium 199 without hypoxanthine or
xanthine and supplemented with 5 mM potassium phosphate, pH
7.4. Cells were then washed three times with HBSS and solubilized
immediately in 3 ml of 1 mM phenylmethylsulfonyl fluoride,
0.5 µg/ml leupeptin, 0.1% CHAPS, 0.1 mM EDTA, 10 mM dithiothreitol, and 50 mM potassium
phosphate, pH 7.4 (lysis buffer). To measure extents of XO inactivation
and/or release, cell monolayers were incubated with XO, washed three
times, and further incubated at 37 °C for 0-6 h with 15 ml of
M199(
). At intervals, the medium was collected, cells were
solubilized with lysis buffer, and both cell-bound and
medium-associated XO activity were measured fluorometrically at
37 °C via oxidation of pterin to isoxanthopterin, using methylene blue as an electron acceptor rather than NAD+ (23). One
unit of XO activity was defined as the amount of enzyme required to
produce 1 nmol of isoxanthopterin/min, while total XOR activity was
defined as the amount of enzyme required to produce 1 nmol of
isoxanthopterin/min in the presence of 10 µM methylene blue.
For determining XO binding affinity and specificity, cell-XO
incubations were conducted at 4 °C to minimize endocytosis. It was
initially determined by treating cells for various times with 1 µg/ml
125I-XO that maximal binding occurred at 5 h. Parallel
incubations with additional catalytically inactivated, nonradioactive
XO (1 mg/ml) allowed correction for nonspecific binding. Cell binding association constants were analyzed with a nonlinear regression curve
fitting program for a single ligand binding model (Enzfitter, Elsevier-BIOSOFT, UK). The influence of BSA, heparin, and pH on XO
binding to cells was assessed using cells treated with
125I-XO in M199(
) at 4 °C in 12-well plates. Cells
were incubated with 0.5 ml of 10 µg/ml 125I-XO in the
absence or presence of 1% BSA or 15 units/ml heparin for 6 h,
after which the cells were washed three times in cold HBSS and
solubilized in lysis buffer. The pH profile of XO binding to cells at
pH 6.8-8.0 was examined by adding 2 µg/ml 125I-XO to
cells at 4 °C as above, except PBS was used for all steps to
optimize pH control.
To ascertain reversibility of XO binding and time-dependent
incorporation of XO into a trypsin-resistant compartment, 1 µg/ml 125I-XO in 0.7 ml of M199(
) was added to cells in
2.2-cm2 12-well plates for 0-70 min at 37 °C. Cells
were washed at intervals with cold Ca2+-,
Mg2+-free HBSS prior to treatment with 0.5% trypsin for 15 min at 37 °C. It was previously determined, from analysis of the
release of [14C]adenine from prelabeled cells, that
trypsin incubations of 15 min or less did not lyse cells. Trypsinized
cells were then centrifuged, and the 125I-XO-associated
radioactivity of cell pellets and the supernatants collected from
trypsinized cell pellets was measured by
-counting. To ensure that
the radioactivity measured was not free 125I dissociated
from XO, both cell lysates and media were made 1% with BSA, and total
protein was precipitated with 10% trichloroacetic acid (w/v) to
determine proportions of acid-soluble and insoluble 125I.
To ascertain the latency of cell-bound XO release or degradation, cells
were incubated with 5 milliunits/ml XO for 1.5 h at 37 °C or 1 µg/ml 125I-XO at 4 °C in M199. Cells were then washed
and postincubated with M199 at 37 °C for up to 6 h, and both
cells and media were obtained at intervals for XO activity assay or
125I-XO protein content determination.
The ability of various compounds to displace cell-bound XO was
determined by incubating cells grown in 12-well plates with 1 µg/ml
125I-XO for 3 h at 4 °C, after which cells were
washed twice with 4 °C HBSS and postincubated for 1 h at
4 °C with gentle shaking in 0.7 ml of M199(
) containing no
additions (control cells), 50 units/ml EC-SOD, 1 or 100 µg/ml
poly-D-lysine, 15-100 units/ml heparin (176 units/mg),
2.5% BSA, or 2.5% bovine calf serum. Cells were then washed with cold
HBSS and solubilized with lysis buffer, and cell-associated
radioactivity was determined. Cells were also pretreated for 3 h
at 37 °C with 4 milliunits/ml heparinase, heparitinase, or
chondroitinase AC in M199 prior to the addition of 125I-XO.
The activity of endoglycosidases under these conditions was determined
spectrophotometrically (
232 = 3800 M
1 cm
1) using 100 µg/ml
heparin, heparan sulfate, or chondroitin sulfate as substrate (24).
After endoglycosidase treatment, cells were washed with HBSS and
incubated with 1 µg/ml 125I-XO in M199(
) for 3 h
at 4 °C. Cells were then washed three times with cold HBSS and
solubilized with lysis buffer. Cell-associated 125I-XO was
then compared with that of parallel control cells not treated with
endoglycosidases. Potential protease contamination of chondroitinase AC
was ruled out using azocasein and fluorescamine-casein as substrates,
with the assay having a lower limit of detection of 10
5% contamination.
XO Immunolocalization--
Polyclonal rabbit anti-XOR was used
for immunolocalization studies. Bovine aortic endothelial cells were
grown to confluency on two-well chamber slides (Nalge-Nunc
International, Milwaukee, WI) and incubated for 1 or 3 h with 5 milliunits/ml XO in M199 at 37 °C. Primaquine (10 mM)
was added in some cases to inhibit endocytosis. Cells were washed with
0.15 M NaCl, 10 mM potassium phosphate, pH 7.4 (PBS) and fixed with 4% paraformaldehyde in PBS for 20 min at
25 °C. Fixed cells were blocked for 15 min with 50 mM
lysine in PBS followed by the addition of 10% goat serum in PBS for
1 h at 25 °C. Permeabilized cell groups had 0.1% Triton X-100
added to both lysine and goat serum-containing blocking buffers. Cells
were then incubated 12-15 h at 4 °C with polyclonal rabbit anti-XOR
antibody in PBS containing 10% goat serum, followed by 30-min
incubation at 22 °C with fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove PA).
Incubation with the secondary antibody was terminated by extensive
washing and 5-min postfixation with 4% paraformaldehyde in PBS. Nuclei
were counterstained by incubation for 15 min with 1 µg/ml
4,6-diamino-2-phenylindole in deionized H2O. Stained cells were mounted with Prolong mounting medium (Molecular Probes, Inc., Eugene, OR). Immunolocalization of added XO and endogenous XOR was
performed using an Olympus IX-70 inverted microscope with an Olympix
digital cooled camera, and images were analyzed using Esprit software
(Life Sciences Resources, Cambridge, UK). The same exposure times and
sensitivity settings were used in the capture and processing of all
digital images. Antibody specificity was determined by incubating the
primary antiserum with 3.6 µg/ml bovine XO overnight at 4 °C
before addition to fixed cell monolayers.
Extracellular Superoxide Release and Xanthine Oxidase Kinetic
Constants--
Superoxide production by cell monolayers was measured
spectroscopically in HBSS supplemented with 10 mM potassium
phosphate, 0.1 mM horse heart ferricytochrome c,
100 µM xanthine, pH 7.4 (HBSS-cyt c) at 550 nm
(
= 21 mM
1 cm
1). Xanthine
oxidase was bound to cells in 25-cm2 flasks by incubation
with 5 ml of 0-10 milliunits/ml XO in M199(
) for 3 h at
37 °C, followed by extensive washing with HBSS. Cell monolayers were
then incubated at 37 °C for 3 h in 3 ml of HBSS-cyt c with or without other additions. A 1-ml aliquot of medium
was removed every 10 min for absorbance measurement and then returned to flasks. The rate of xanthine or pterin consumption by bound XO was
similarly determined by measuring production of uric acid or
isoxanthopterin. Xanthine or pterin-containing HBSS was added to cells,
and a 1-ml aliquot of medium was removed every 10 min for
spectrophotometric determination of xanthine oxidation to urate
(
295 = 1.1 × 104
M
1 cm
1) or fluorometric
analysis of rates of isoxanthopterin oxidation to pterin
(isoxanthopterin;
ex = 340 nm,
em = 395 nm).
[14C]Adenine Release--
Cells grown in 12-well
plates were incubated with 4 µM (0.2 µCi/ml)
[14C]adenine for 3 h in HBSS at 37 °C. During the
last 2 h of [14C]adenine labeling, XO was added,
followed by extensive washing with HBSS. Following an 8-h
postincubation of washed and radiolabeled cells in HBSS supplemented
with 0.1 mM xanthine, aliquots of media were removed, and
cell lysis was determined by [14C]adenine and
[14C]adenine-labeled metabolite release into the medium.
The extent of [14C]adenine release from cells solubilized
with 1% CHAPS indicated 100% lysis.
Smooth Muscle Cell cGMP Measurement--
Endothelial cells grown
to confluence on 2.2-cm2 Transwell micropore filters were
incubated with 10 milliunits/ml XO in M199(
) for 3 h at
37 °C, followed by extensive washing with HBSS. Endothelial cell-containing filters were then placed in six-well plates containing confluent rat aortic smooth muscle cells and equilibrated for 10 min
with Dulbecco's PBS. Dulbecco's PBS supplemented with 100 µM xanthine, 50 units/ml CuZn-SOD, and 0.5 mM
isobutylmethylxanthine with or without 100 µM allopurinol
was then added to smooth muscle cells, while Dulbecco's PBS with 100 µM xanthine, 50 units/ml SOD, 0.5 mM
isobutylmethylxanthine, 6.7 µM ionomycin with or without 100 µM allopurinol was added to overlaid endothelial
cell-containing Transwell filter wells for 15 min. A low concentration
of CuZn-SOD (5 units/ml) was added to all groups to scavenge variable
rates of light-induced O
2 production in the medium induced by
intermittent exposure to dimmed laboratory lighting conditions and
autoxidation of medium constituents. The basal generation of
O
2 by medium under these conditions was confirmed via
chemiluminescent assay of accelerated rates of ·NO decay when
medium was exposed to laboratory lighting conditions, as opposed to
slower rates of ·NO decay in darkness or in the presence of 5 units/ml SOD. Sodium nitroprusside (10 µM) was added to
preparations of smooth muscle cells not exposed to endothelium-derived
mediators to determine maximal smooth muscle cell cGMP production. In
some cases, heparin-binding EC-SOD (50 units/ml) or higher
concentrations of CuZn-SOD (1000 units/ml) was added to the media of
both the effector (endothelial) and target (smooth muscle) cells during
the the 15-min exposure period. Endothelial cell-containing Transwell
inserts were then removed from smooth muscle cell-containing wells, and
0.5 ml of cold 50 mM sodium acetate, pH 4, was added to
smooth muscle cells for 1 h at 4 °C. Cells were then removed by
scraping and centrifuged at 2000 × g for 5 min, and
the supernatant was assayed for cGMP by EIA. Smooth muscle cells were
not morphologically affected by any of the above manipulations. For all
experiments, protein concentrations were measured at 550 nm by a
modified Bradford assay using Coomassie Plus reagent (Pierce) with
bovine serum albumin as a standard (25). Samples were diluted with
water to 5-20 µg/ml to which an equal volume of Coomassie Plus
reagent was added.
Statistical Analysis--
Data were analyzed with a nonlinear
regression curve-fitting program (Enzfitter, Elsevier-Biosoft, UK)
using a Michaelis-Menten equation to determine kinetic constants.
Unless otherwise noted, data represent mean ± S.D. for
experiments repeated at least two times, all giving similar results.
Statistical significance of p < 0.05 was determined by
one-way analysis of variance with Duncan's post hoc analysis.
 |
RESULTS |
Xanthine Oxidase Binding to Endothelial Cells--
Initial
endothelial cell 125I-XO binding studies were conducted at
4 °C to minimize possible endocytosis and proteolysis. Maximum binding occurred by 5 h (mean kon = 0.39 ± 0.21 µg h
1) and was resistant to removal
by extensive washing. Xanthine oxidase binding was saturable,
exhibiting an average Kd of 0.9 µg/ml (6 nM XO, Fig. 1), with
experiment to experiment Kd values ranging from 0.3 to 2.2 µg/ml (2-15 nM XO) for different bovine
endothelial cell lines. Saturable binding was 80-90% inhibitable by
the addition of a 100-fold excess of unlabeled XO. The addition of 15 units/ml (85 µg/ml) heparin during XO-cell incubation (2 µg/ml XO)
inhibited binding 43%, while BSA (100 µg/ml) had no effect (not
shown). Pretreatment of cells with heparitinase or heparinase did not
affect subsequent XO binding, while pretreatment with chondroitinase AC
(determined to have no detectable contaminating protease activity)
inhibited XO binding 47% (Table I).
Alkaline pH enhanced binding of XO to cells 4.3-fold at pH 8, compared
with pH 7.4 (Table II).
Poly-D-lysine and heparin, added for 1 h at 4 °C,
following binding of XO to cells, displaced XO 45 and 26%,
respectively, compared with buffer-treated controls (Table
III). Heparin-binding EC-SOD, BSA, and
fetal bovine serum did not displace cell-bound XO.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Specific and nonspecific binding of xanthine
oxidase to endothelial cells. Cells were incubated with increasing
concentrations of 125I-XO at 4 °C for 6 h in the
absence ( ) or presence ( ) of 1 mg/ml native XO. Data represent
mean ± S.D. of a representative experiment (n = 6).
|
|
View this table:
[in this window]
[in a new window]
|
Table I
The role of endoglycosidases in endothelial cell-xanthine oxidase
binding
Cells were treated with glycosaminoglycan lyases at 37 °C for 3 h prior to incubation with 1 µg/ml 125I-XO at 4 °C for
3 h. Data represent mean ± S.D. of three experiments
(n = 4).
|
|
View this table:
[in this window]
[in a new window]
|
Table II
The influence of pH on endothelial cell-xanthine oxidase binding
Cells were incubated with 2 µg/ml 125I-XO in PBS at various
pH values for 6 h at 4 °C. Cells were washed with PBS (adjusted
to the same pH) three times and solubilized, and cell-bound
125I-XO was determined. Data represent mean ± S.D. of
three separate experiments, n = 12 for each.
|
|
View this table:
[in this window]
[in a new window]
|
Table III
Displacement of endothelial cell-bound xanthine oxidase
Cells were incubated at 4 °C with 1 µg/ml 125I-XO in M199
without hypoxanthine or xanthine for 3 h. Cells were washed and
incubated with M199 without hypoxanthine or xanthine in the absence
(control cells) or presence of the noted additions for 1 h at
4 °C. The concentration of 125I-XO associated with cells was
then measured. Data represent mean ± S.D. of two experiments,
n = 6.
|
|
XO Binding at 37 °C--
Endothelial cells bound XO at 37 °C
in a time- and concentration-dependent manner. The
cell-associated XO remained active, with exposure to 2.5 milliunits/ml
XO or greater resulting in significant increases in cell XO catalytic
activity (Fig. 2). At 37 °C, maximum
cellular XO catalytic activity and 125I-XO binding occurred
within 1 h of XO addition (kon = 5.5 ± 1.2 h
1). Endothelial XDH, as well as XO, activity
increased after XO incubation with cells, such that the proportion of
XO activity compared with total xanthine oxidoreductase activity
remained virtually unchanged (34 ± 4% for control cells, 39 ± 3% for cells incubated with 5 milliunits/ml XO; data not shown).
The possible endocytosis of cell-bound XO during cell exposure to XO at
37 °C was probed using 0.5% trypsin to remove cell
surface-associated proteins. Cells were exposed to trypsin for the
maximal time possible before disruption of cell membrane integrity
became apparent. There was an initial rapid increase, followed by a
slower increase, in the pool of trypsin-resistant cell-associated XO,
implying ongoing endocytosis of XO while binding to cells (Fig.
3).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Xanthine oxidase binding increases
endothelial cell xanthine oxidase specific activity and rates of
superoxide production. Xanthine oxidase (0-10 milliunits/ml) was
incubated with the cells for 3 h at 37 °C in M199 without
hypoxanthine or xanthine, after which the cells were washed and either
assayed for XO activity via fluorometric analysis of pterin oxidation
( ) or incubated with HBSS supplemented with 0.1 mM
cytochrome c and 0.1 mM xanthine for determining
rates of O 2 production ( ). Reduction of cytochrome
c was monitored at 550 nm for 2-3 h. Data represent
mean ± S.D., n = 3 (cytochrome c
reduction) and n = 4 (XO activity) of a representative
experiment. *, p < 0.05 compared with no addition of
XO.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Time-dependent endocytosis of
cell-bound xanthine oxidase. Cells were incubated with 1 µg/ml
125I-XO in M199 at 37 °C in the absence of added
substrate for 0-70 min, washed, and treated with 0.5% trypsin.
Trypsinized cells were centrifuged, and 125I-XO was
measured in both trypsin-treated cell pellets ( ) and supernatants of
trypsinized and centrifuged cells ( ). Data represent the mean ± S.D. of a representative experiment (n = 4).
|
|
Further evidence of bound XO endocytosis by endothelial cells was
observed immunocytochemically with anti-XO antibodies (Fig. 4). Endothelial cells exposed to 5 milliunits/ml XO at 37 °C demonstrated increasing amounts of XO on
the cell surface, while unexposed cells had no XO on the cell surface.
When cells were permeabilized to immunolocalize both intra- and
extracellular XO, unexposed cells displayed punctate staining,
suggesting that intracellular XO was contained in organelles, possibly
endosomes. Cells exposed to XO and then permeabilized had a greater
degree of intracellular staining than unexposed cells. The addition of
the endocytosis inhibitor primaquine to cells slightly increased
extracellular XO immunostaining and inhibited the increase in
intracellular staining of XO in cells exposed to XO. No fluorescence
was detected when using primary antiserum preadsorbed with 5 milliunits/ml purified bovine XO.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 4.
Immunolocalization of the distribution of
endogenous and added exogenous xanthine oxidase in vascular endothelial
cells. Cells were incubated with 5 milliunits/ml XO in M199 at
37 °C for 1 or 3 h and washed extensively. Primaquine was added
to the XO + inh group during XO incubation to limit endocytosis.
Control and treated cells were then fixed with paraformaldehyde and
blocked in the presence or absence of 0.1% Triton X-100 to
permeabilize the cell surface. The presence of XO was then probed at
4 °C using rabbit polyclonal anti-XDH/XO antibody and fluorescein
isothiocyanate-conjugated secondary antibody. The nuclei were
counterstained with 4,6-diamino-2-phenylindole. Scale
bar, 50 µm.
|
|
After XO was bound to cells at 37 °C, changes in cell-associated XO
activity (Fig. 5A) and
trypsin-dissociable 125I-XO (Fig. 5B) were
examined over time. Augmented cell XO activity and cell-associated
125I-XO declined with half-lives of 1.8 and 2.8 h,
respectively. All of the 125I-XO released from cells was
detected in the media as trichloroacetic acid-precipitable
125I-labeled protein. Part of the XO specific activity lost
by cells was recovered as intact active enzyme in the media within the first hour, after which there was no significant increase in medium XO
activity, despite a continued loss of cell-associated XO activity. All
of the activity detected in the medium was XO in the oxidase rather
than dehydrogenase form.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Latency of endothelial cell-bound xanthine
oxidase. Cells were incubated with 5 milliunits/ml XO at 37 °C
(A) or 1 µg/ml 125I-XO at 4 °C
(B) in M199. Cells were then washed and further incubated
with M199 for various times before assessing the XO activity or
125I-XO protein associated with cells ( ) and media
( ). Data represent the mean ± S.D. of a representative
experiment (n = 4).
|
|
Superoxide Production by Cell-associated XO--
Increased
cell-bound XO activity, following incubation with 0-10 milliunits/ml
XO for 3 h, was paralleled by increased rates of cytochrome
c reduction in the presence of xanthine (Fig. 2). The rate
of cytochrome c reduction by cells having bound XO was inhibited 74% by allopurinol, 17-29% by 300 units/ml CuZn-SOD, polyethylene glycol-conjugated SOD, or Mn-SOD and 41% by 50 units/ml EC-SOD (Table IV). When succinoylated
cytochrome c was used to detect O
2 rather than
native cytochrome c, CuZn-SOD completely inhibited
cytochrome c reduction (Table IV). There was no detectable cytochrome c reduction induced by the media during analysis
of extracellular O
2 production by cells with bound XO, nor was
there significant SOD-inhibitable cytochrome c reduction by
cells prior to XO binding.
View this table:
[in this window]
[in a new window]
|
Table IV
Inhibition of endothelial cell monolayer cytochrome c reduction by
superoxide dismutases and allopurinol
Cells were incubated with or without XO for 3 h at 37 °C.
Monolayers were then washed three times and incubated with 0.1 mM native or succinoylated ferricytochrome c,
0.1 mM xanthine, and bovine CuZn-SOD,
polyethyleneglycol-conjugated bovine CuZn-SOD (PEG-SOD), human Mn-SOD,
EC-SOD, or allopurinol. Data represent the mean ± S.D. of two
separate experiments (n = 3).
|
|
The Km for oxidation of xanthine and pterin by
cell-associated XO was determined in intact cell monolayers. The
Km for xanthine oxidation, indicated by rates of
uric acid production, was 3.4 ± 0.8 µM for
cell-bound XO and separately, 3.6 ± 0.5 µM for
soluble XO. The Km for pterin oxidation by
cell-bound XO, measured fluorometrically by isoxanthopterin formation,
was 1.5 ± 0.3 µM for cell-bound XO and, separately,
1.6 ± 0.8 µM for soluble XO (Table
V). Minimal xanthine and pterin oxidation
was due to cell-associated XO being released into the media during analysis. Both xanthine and pterin oxidation was fully
allopurinol-inhibitable. Comparison of rates of xanthine oxidation and
cytochrome c reduction at similar xanthine concentrations
indicated that 22 ± 6% of the electron flux to oxygen by bound
XO was univalent, yielding O
2.
View this table:
[in this window]
[in a new window]
|
Table V
Kinetic constants of xanthine oxidase in solution and following
endothelial cell binding
Cells were incubated with 5 milliunits/ml XO for 3 h at 37 °C.
Monolayers were washed, and rates of substrate oxidation were
determined for 0.02-20 µM xanthine or pterin. Similar
analyses were conducted for native bovine XO free in solution.
|
|
Binding of XO to Other Cell Types--
The binding of 5 milliunits/ml XO to cultured human umbilical vein endothelium, fetal
rat lung fibroblasts, and fetal rat lung type 2 epithelial cells also
resulted in significant increases in cell XO specific activity. This
yielded now-detectable levels of XO in human vascular endothelium,
fibroblasts, and type 2 cells as well as a 7-fold increase in bovine
aortic endothelial cell XO specific activity (Table
VI). Cell-XO binding was dose- and time-dependent, being saturable for epithelial cells but
not fibroblasts (not shown).
View this table:
[in this window]
[in a new window]
|
Table VI
Xanthine oxidase binding to vascular endothelial and pulmonary cells
Cells were incubated with 5 milliunits/ml XO in HBSS at 37 °C for
3 h prior to washing and assay of cell-associated XO activity by
pterin oxidation to isoxanthopterin. Data represent mean ± S.D.
of a representative experiment (n = 3-5).
|
|
Influence of Bound XO on Endothelial Function--
To determine
whether cell-bound XO influenced endothelial function, endothelial
cells were incubated with XO at 37 °C, washed, and then further
incubated with 100 µM xanthine. After exposure to
xanthine for up to 8 h, it was observed that cellular membrane integrity was not significantly impaired, as indicated by release of
[14C]adenine and labeled cell metabolites into the media
(>80% trichloroacetic acid-soluble; data not shown). Cell-bound XO,
in the presence of xanthine, interrupted ionomycin-stimulated
endothelial ·NO-dependent signaling to smooth muscle
cells, as revealed by a significant reduction in endothelial-stimulated
cGMP production by smooth muscle cells treated with the
phosphodiesterase inhibitor isobutylmethylxanthine (Fig.
6A). Inactivation of XO by
pretreatment with KCN or the addition of allopurinol to incubations
abrogated the inhibitory action of XO toward
·NO-dependent signaling. The addition of 1000 units/ml CuZn-SOD or 50 units/ml EC-SOD to ionomycin-stimulated
endothelial cells having bound XO did not restore smooth muscle cell
guanylate cyclase activation (Fig. 6B).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
A, inhibition of nitric
oxide-dependent signal transduction by cell-bound xanthine
oxidase. Vascular endothelial cells (EC) cultured on
Transwell filters were incubated with 10 milliunits/ml XO for 3 h
and washed extensively. Endothelial cell-containing filters were then
transferred to dishes containing smooth muscle cells and incubated with
xanthine (100 µM), SOD (50 units/ml),
isobutylmethylxanthine (0.5 mM), and ionomycin (6.7 µM) with or without allopurinol for 15 min. Smooth muscle
cell cGMP concentration was then determined by EIA. Sodium
nitroprusside (10 µM) was added directly to smooth muscle
cells to reflect maximum attainable cGMP concentrations. Data represent
mean ± S.E. of six experiments (n = 3). *,
p < 0.05 compared with ionomycin-treated endothelial
cells. B, inability of EC-SOD or CuZn-SOD to inhibit
cell-bound xanthine oxidase-mediated impairment of nitric
oxide-dependent signaling. Vascular endothelial cells and
smooth muscle cells were treated as described above with the further
addition of 50 units/ml EC-SOD or 950 units/ml CuZn-SOD to the media of
both cell types during the 15-min coincubation period. Data represent
mean ± S.E. of a representative experiment (n = 3).
|
|
 |
DISCUSSION |
The observation that XO binds to endothelial cells (11, 14) has
been extended to define the affinity and nature of cell binding,
retention of catalytic activity by cell-associated XO, the endocytosis
of cell-bound XO, and finally, the cellular structural and functional
consequences of a concentrated production of reactive species at sites
of XO-cell association. The specific and reversible binding of XO by
endothelial cells was demonstrated by saturable binding at 4 °C
(Fig. 1) and 80-90% inhibition of 125I-XO binding by a
100-fold excess of unlabeled XO. The latter also confirmed that
iodination did not significantly modify XO-cell interactions. Binding
was not inhibited by albumin and serum, indicating the potential for
in vivo occurrence as well as the absence of nonspecific XO
binding to cell surface or culture dish sites (Table III). The
Kd of XO binding (6 nM) to endothelial cells is comparable with other macromolecules having specific endothelial binding sites, including lipoprotein lipase, diamine oxidase, and EC-SOD (26-28). Early evidence that exogenous XO could bind to and damage endothelial cells came from the observations that
the addition of heparin to XO-treated cells partially limited oxidative
injury by mechanisms that did not include the direct scavenging of
reactive species by heparin (29, 30). Previous studies supported the
concept that the endogenous XOR content of non-rodent vascular cells is
too low in specific activity to contribute significantly to oxidative
signal transduction events or cell injury (31, 32). The capacity of the
splanchnic system and both normoxic and hypoxic vascular endothelial
cells to express and release XOR into the circulation affirm that
extracellular XO has the potential to contribute to tissue pathogenesis
(13, 33-35).
There are undetectable or only trace levels of XO activity in normal
human plasma (36, 37), with up to 3% of total IgM being anti-human
XOR, existing predominantly as immune complexes with endogenous XOR
(38). During pathological conditions such as reperfusion injury,
hepatitis, adult respiratory distress syndrome, or atherosclerosis,
human plasma XO concentrations increase to as much as 1.5 milliunits/ml, an amount of XO that would occupy a significant fraction
of cellular binding sites, assuming a specific XO activity of 1.4 units/mg of protein (12, 36, 37). These elevated XO levels can persist
for at least 120 min following reperfusion of ischemic tissue, a time
frame permitting maximal target cell binding to occur (Fig. 3; Refs.
11, 12, and 40). Normal levels of plasma purine substrates for XO
(e.g. hypoxanthine) are 1-2 µM, increasing up
to 15 µM during anaerobic exercise and in the plasma of
patients having chronic impaired lung function (e.g.
neonatal and adult respiratory distress syndromes). Also, immediately
after reperfusion of ischemic tissues, plasma purine concentrations can
rise to over 100 µM (41-44). Thus, circulating XO can
bind in the vascular compartment of humans, utilize endogenous purine
substrates to generate reactive species, and impair vascular function.
This concept is supported by recent observations in both animal models
and clinical studies of ischemia-reperfusion, hypertension, and
atherosclerosis (45-48). For example, the addition of heparin to the
vascular compartment of normal humans and rat hemorrhagic shock models
causes an immediate increase in plasma XO content in the absence of
additional acute cell injury, suggesting reversible binding of XO to
the vascular lumen (11, 14). Also, isolated lungs perfused with XO-rich
effluent from ischemic rat livers exhibit a 1.4-fold increase in whole
lung XO activity (13). Rabbit aortic rings exposed to XO displayed
heparin and allopurinol-reversible impairment of
endothelial-dependent relaxation, underscoring the adverse
functional consequences of cell-XO association (45). It is noteworthy
that the calf serum utilized in tissue culture systems is rich in XOR.
Thus, cell binding of this frequent culture medium component can
contribute to net cell XO specific activity, changes in cell XO
distribution, and an elevation in endogenous rates of cell oxidant
production (49).
Although the endothelial XO binding site has not been identified at a
molecular level, its functional characteristics are revealed herein.
The nonlinearity of Scatchard binding analysis and the variability in
XO binding constants for different passages and lines of cells suggests
heterogeneity in XO binding sites. Some heparin-binding molecules
(e.g. thrombospondin, EC-SOD, and basic fibroblast growth
factor) are >80-90% inhibited from cell binding by low
concentrations (1-10 µg/ml) of heparin or after cellular treatment
with heparin lyases (50-52). Derivatization of lysine and arginine
residues of XO limited binding to heparin-Sepharose conjugates (53).
Herein, the addition of high heparin concentrations and hydrolysis of
heparin and chondroitin sulfate-containing proteoglycans prior to or
after endothelial cell incubation with XO only partially inhibited XO
binding, suggesting heterogeneous binding sites that include
chondroitin sulfate-containing proteoglycans (Tables I and III). The
partial displacement of XO binding by heparin may be due to the
polyanionic character of heparin and its ability to bind to cationic
motifs of XO, in turn competing for XO binding to chondroitin sulfate
or other glycosaminoglycan-containing cellular proteoglycans. Evidence
that XO does not exclusively bind to heparin-containing cell
proteoglycans comes from comparing XO kinetic properties following
binding to heparin-Sepharose and to endothelial cells. Xanthine oxidase
immobilized by heparin-Sepharose had an increased Km
for xanthine and an increased proportion of univalent electron flux
(15). In contrast, cell-bound XO manifested the same
Km for xanthine and percentage of univalent flux as
XO in solution. In addition, XO binding to heparin-Sepharose increased
with acidic pH, while XO binding to cells increased with alkaline pH
(Table II; Ref. 15).
The association of XO with cells led to increased endothelial XO
activity and content of 125I-labeled XO (Figs. 2 and 3).
Cell-bound XO became incorporated by endocytosis, as indicated by the
temporally increasing proportion of cell-associated XO resistant to
trypsin dissociation (Fig. 3) and the diminished intracellular XO
immunoreactivity of XO-treated cells when coincubated with the
endocytosis inhibitor primaquine (Fig. 4). The decline of
cell-associated XO specific activity, coupled with changes in cell
versus medium distribution of both catalytically active XO
and 125I-XO, revealed that cell-bound XO was also being
both inactivated and rereleased from cells (Fig. 5), similar to the
fates of EC-SOD and lipoprotein lipase (26-28). The reversibly bound
XO found in media that retained activity did not reveal the modest
(~20%) increased rates of substrate oxidation upon the addition of
methylene blue observed for control preparations of XO (not shown).
Methylene blue directly oxidizes the FeS centers of XO, unlike oxygen,
which oxidizes the FAD cofactor (54). The lack of an influence of methylene blue on rate indicates that cell-released XO utilized oxygen
as an electron acceptor, while the original preparation of XO contained
partial dehydrogenase activity. This, coupled with the observation that
the addition of XOR to fresh plasma immediately converts from the
dehydrogenase to oxidase form (11, 55), implies that both release of
XOR from tissues into the circulation and binding of XOR to cells will
favor the production of reactive oxygen species. Since XOR also
displays significant NADH oxidase activity, the cell-bound form may be
one of multiple loci for the extracellular
NAD(P)H-dependent production of O
2 by vascular
tissues (45, 56-58).
Superoxide generated by cell-bound XO was resistant to scavenging by
CuZn-SOD, a phenomenon also observed for heparin-Sepharose 4B-immobilized XO (Table IV; Ref. 15). Cytochrome c
reduction by XO in solution was completely inhibited by 50 milliunits/ml CuZn-SOD, while cytochrome c reduction by
cell-associated XO was only inhibited 28-40% by 300 milliunits/ml
CuZn-SOD, Mn-SOD, PEG-derivatized CuZn-SOD, and 50 units/ml EC-SOD.
There was, however, a significant difference in the extent of
inhibition of native cytochrome c reduction by CuZn-SOD
(28% of control), compared with the heparin-binding EC-SOD (40% of
control). This suggested that SOD-resistant, XO-dependent cytochrome c reduction arises from partitioning of
XO-derived O
2 production into an anionic compartment.
Alternatively, a component of SOD-resistant XO-dependent
cytochrome c reduction could be the result of direct
cytochrome c reduction, since proteolyzed XO can directly
reduce cytochrome c (28). To address this issue, it is noted
that native cytochrome c at pH 7.4 bears a +9.5 charge, thus
avidly associating with the glycocalyx, while CuZn-SOD is electrostatically repelled by cells (59). To reveal whether cell-bound
XO reduced O2 to O
2 or directly reduced cytochrome c, cytochrome c was succinoylated to lend a net
negative charge at pH 7.4 and limit association with the glycocalyx and
cell-bound XO. The net negative charge on succinoylated cytochrome
c also decreases the rate constant for reduction by
O
2, making it necessary to use greater concentrations of
succinoylated cytochrome c to achieve the same rate of
reduction as native cytochrome c (22). The complete
inhibition of cell-associated XO-dependent succinoylated cytochrome c reduction by CuZn-SOD confirmed the production
of O
2 by cell-bound XO rather than direct electron transfer to
cytochrome c (Table IV). The inability of native CuZn-SOD to
inhibit O
2 production by cell-bound XO, due to formation of
O
2 in a sequestered microenvironment, has important
implications for devising antioxidant strategies for treatment of
diseases including a pathogenic role for XO-derived reactive oxygen
species and other cellular sources of O
2. The data herein
reveal that XO inactivation, inhibition of cell-XO binding, and the
administration of proteoglycan-binding forms of SOD or more cell-avid
SOD mimetics will have greater pharmacologic impact on this reaction
pathway than native CuZn-SOD (60, 61).
Evidence that cell-bound XO can impair vascular cell function and
produces O
2 in a sequestered microenvironment comes from the
inhibition of ·NO-dependent signal transduction by
endothelial cells having bound XO (Fig. 6) Stimulation of control
endothelial cell ·NO production by ionomycin increased smooth
muscle cell cGMP production in co-cultures of endothelial and smooth
muscle cells as before (62, 63). Superoxide produced by cell-bound XO
can then react with ·NO at rates 10-fold faster than for SOD
scavenging of O
2, yielding ONOO
and
attenuating ·NO-dependent production of cGMP by
smooth muscle cells (58, 64-66). Peroxynitrite generated from the
reaction of XO-derived O
2 with ·NO was not
expected to directly inhibit endothelial nitric-oxide synthase (67).
The prevention of XO-mediated inhibition of
endothelial-dependent smooth muscle cell cGMP production by
allopurinol indicated that XO did not irreversibly inhibit nitric-oxide
synthase activity or deplete cellular substrates required for
nitric-oxide synthase activity (Fig. 6). The lack of an
effect of CuZn-SOD or EC-SOD on XO-mediated inhibition of smooth muscle
cell cGMP production also reinforced the concept that
glycosaminoglycan-bound XO partitions into an SOD-resistant
compartment (15).
Multiple lines of evidence reveal that circulating and cell-associated
XO contribute to the pathogenesis of vascular disease. Patients with
atherosclerosis that display impaired vascular function also exhibit
increased circulating XO levels, a phenomenon that may predispose to
impaired ·NO-dependent signal transduction and
further development of arterial lesions via oxidative modification of
low density lipoproteins and depletion of plasma antioxidants (39).
While XO can be detected in a heparin-reversible vascular compartment
in healthy adults (14), increased levels of XO are also evident in the
vessel wall and plaque of individuals with atherosclerosis (68). This increase in vessel wall XO may come from enhanced expression of XO by
local stimuli (69-71) as well as from binding of plasma XO to the
endothelium and intima. Then vessel wall XO can initiate a cascade of
oxidant-mediated events that culminate in a chronic inflammatory
response, promotion of cellular destruction, and plaque formation (58,
72-75). Importantly, endothelial-bound XO inhibits
·NO-dependent signal transduction events that
mediate vessel relaxation (Fig. 6). This phenomenon, a hallmark of
vascular pathology in both animal models of atherosclerosis and
atherosclerotic humans, is amenable to normalization by allopurinol
(45, 46, 76).
In summary, diverse cell types possess glycosaminoglycan-containing
binding sites for XO. Upon cell binding, XO retains catalytic activity
and becomes incorporated via endocytosis. The reactive oxygen species
derived from cell-bound XO are generated in an SOD-resistant
compartment to inhibit ·NO-dependent signaling and
initiate proinflammatory events.