© 1999 American Heart Association, Inc.
Original Contribution |
Opposing Effects of Reactive Oxygen Species and Cholesterol on Endothelial Nitric Oxide Synthase and Endothelial Cell Caveolae
From the Department of Medicine (T.E.P., V.P., H.U., A.W.), Division of Cardiology, University of Washington, Seattle, Wash, and the Center for Cardiovascular Research (C.Y., B.C.B.), University of Rochester, Rochester, NY. The current affiliation for T.E.P. is the Department of Cardiovascular Diseases and the Molecular Medicine Program, Mayo Clinic, Rochester, Minn.
Correspondence to Bradford C. Berk, MD, PhD, University of Rochester Medical Center, Center for Cardiovascular Research, 601 Elmwood Ave, Box 679, Rochester, NY 14642. E-mail bradford_berk{at}urmc.rochester.edu
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Abstract |
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Abstract—Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 µg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 µmol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.
Key Words: superoxide • caveolae • caveolin • eNOS • cholesterol
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Introduction |
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In cardiovascular diseases such as atherosclerosis, hypertension, and diabetes, impaired nitric oxide (NO) bioactivity has been suggested to be pathogenic and to contribute to vascular dysfunction. NO is produced locally by endothelial nitric oxide synthase (eNOS) within the vessel wall. In normal endothelium, eNOS activity (and hence NO production) is acutely regulated by increases in [Ca2+]i,1 2 by agonists such as bradykinin and acetylcholine,3 as well as by hemodynamic stimuli, especially fluid shear stress. eNOS expression is also chronically regulated by several stimuli including fluid shear stress4 5 and transforming growth factor-ß (TGF-ß).6
Recent findings suggest that the subcellular localization of eNOS is another important regulator of NO production.3 7 eNOS subcellular localization appears to be highly regulated in a dynamic manner by multiple mechanisms. (1) N-myristoylation has been shown to be required for eNOS membrane association8 and Golgi binding.9 (2) Palmitoylation is required for eNOS localization to caveolae.10 11 12 13 (3) Protein-protein interactions occur between eNOS and membrane-associated proteins including caveolin,14 heat shock protein 90,15 and G protein–coupled receptors.16 (4) eNOS interactions with anionic membrane phospholipids have also been demonstrated.17 In fact, several groups have proposed that dynamic cycling of eNOS from Golgi to the plasma membrane may regulate eNOS activity.9 12 18
eNOS is typical of a large number of dually acylated proteins that are localized to plasmalemma invaginations called caveolae. Caveolae are specialized membrane domains that have been proposed to play multiple roles in cell function including ion transport, fluid transcytosis, and signal transduction.19 Caveolae have been identified as nonclathrin-coated vesicles highly enriched in sphingomyelin, cholesterol, and the 21-kDa coat protein caveolin. A unique feature of caveolae proteins is relative insolubility in Triton X-100.20 New techniques have been developed to isolate caveolae membranes from other membranes based on their low buoyant density in sedimentation gradients.21 With use of these techniques, cholesterol has been shown to regulate caveolae function, because cells depleted of cholesterol have decreased caveolae numbers.22
The findings that caveolae formation and eNOS subcellular localization are dynamically regulated suggest that pathological states may be associated with disruption of eNOS-caveolae interactions. eNOS appears to associate with caveolae in part by interacting with caveolin. eNOS and caveolin-1 colocalize in the Triton X-100 insoluble fraction of bovine aortic endothelial cells (BAECs),12 and eNOS and caveolin-1 can be coimmunoprecipitated, suggesting a strong interaction.18 In addition, Couet et al23 have identified a highly conserved domain in caveolin-1, termed the caveolin-scaffolding domain that mediates interactions with caveolae-associated proteins. More recently, a motif was identified in several caveolae-associated proteins (including eNOS) that confers binding to the caveolin-scaffolding domain.
We propose that reactive oxygen species (ROS) are powerful perturbants of caveolae structure and eNOS-caveolin interactions. ROS may disrupt eNOS-caveolae interactions by several mechanisms. First, oxidation of cholesterol may alter caveolae structure and cause caveolin to shuttle to Golgi membranes.24 Second, protein-protein interactions such as eNOS-caveolin may be disturbed by oxidation of critical amino acids required for stable interaction or membrane localization. Third, ROS are powerful activators of signal transduction events that may alter eNOS phosphorylation and subcellular localization. In the present study, we show for the first time that cholesterol treatment increases eNOS association with caveolae, that ROS inhibit caveolae formation, and that the association of eNOS with caveolae (and caveolin) is disrupted by ROS.
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Materials and Methods |
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Materials
Anti-eNOS, anti-caveolin, and anti-ERK1/2 antibodies were purchased from Transduction Laboratories. Anti-Src antibody was purchased from Oncogene Science. Anti-actin antibody was purchased from Sigma Chemical Co. LY83583 was purchased from Alexis Biochemicals. Optiprep and Percoll were from Gibco BRL, and xanthine oxidase was from Boehringer Mannheim. Collagenase was purchased from USB Biochemicals, vanadium III chloride from Aldrich Chemicals, and cholesterol, lucigenin (bis-N-methylacridinium nitrate), xanthine, and buffer reagents from Sigma Chemical Co.
Cell Culture and Generation of Superoxide in BAECs With
LY83583
BAECs were isolated and grown in medium 199 (Gibco BRL)
supplemented with 10% FCS (Summit), basal MEM vitamins, and amino
acids (Gibco). BAECs between passage numbers 2 and 8 were grown in
60-mm dishes until confluent. Cholesterol was prepared
fresh before incubation and was added directly to the medium in 100%
ethanol (final concentration <0.1%). As previously
characterized,25 minimal cholesterol oxidation
occurs under these conditions. To generate ROS, the
napthoquinolinedione LY83583 was used as described
previously.26 ROS formation was measured by lucigenin
chemiluminescence26 and by oxidation of
aconitase.27 28 For the aconitase assay, cells were
harvested and centrifuged, and the pellet disrupted in 100 µL
of buffer (containing 50 mmol/L Tris-Cl [pH 7.4], 0.6
mmol/L MnCl2, and 20 µmol/L fluorocitrate)
by applying 10 1-second bursts with a microtip sonic oscillator. The
lysate was centrifuged, and the supernatant assayed for
protein. Aconitase activity was then determined with 10 to 100 µg of
extract protein by following the change in absorbance at 340 nm at
25°C in a 1.0-mL reaction mixture containing 50 mmol/L Tris-Cl
(pH 7.4), 5 mmol/L sodium citrate, 0.6 mmol/L
MnCl2, 0.2 mmol/L
NADP+, and 1 to 2 U of isocitrate dehydrogenase.
This assay is based on the formation of NADPH from
NADP+. A control reaction lacking citrate and
isocitrate dehydrogenase was performed to assay NADPH
production independent of aconitase activity.
Isolation and Characterization of Caveolae From BAECs
Cells were treated for 24 hours with 30 µg/mL
cholesterol or cholesterol plus 1 µmol/L
LY83583. To prepare caveolae,21
50x106 BAECs were harvested and
homogenized in cold buffer A (0.25 mol/L sucrose, 1
mmol/L EDTA, and 20 mmol/L Tricine [pH 7.8]) and
centrifuged at 1000g for 10 minutes. The supernatant
was saved and layered onto 30% Percoll and centrifuged at
84 000g for 30 minutes. The plasma membrane fraction was
collected and brought to a volume of 2 mL with buffer A. An aliquot of
this fraction was saved and assayed as the crude membrane fraction. The
crude membrane fraction was then sonicated 3 times for 30 seconds,
resuspended in a 23% solution of OptiPrep, and then placed in a
centrifuge tube. A linear 20% to 10% OptiPrep gradient was
layered on top and centrifuged at 52 000g for 90
minutes. The top 5 mL of the gradient was collected, resuspended in
50% OptiPrep, and a 15% to 5% OptiPrep gradient was gently layered
on top and centrifuged again at 52 000g for 90
minutes. A distinct opaque band then appeared in the 5% OptiPrep
overlay, which contained caveolae membranes. To characterize
caveolae-associated proteins, proteins were size-fractionated by 9%
SDS-PAGE. Protein concentrations were determined, and equal amounts of
protein were loaded on each lane. After transfer to nitrocellulose
membranes, proteins were stained with Ponceau S to determine equal
protein loading. For Western blot analysis, membranes were
incubated for 1 hour in blocking buffer (Gibco) followed by a 1-hour
incubation with either anti-eNOS or anti–caveolin-1 monoclonal
antibodies. Nitrocellulose membranes were then incubated for 1 hour
with HRP-conjugated anti-mouse IgG antibody and visualized using
enhanced chemiluminescent detection (Amersham). Protein detection by
Western blot analysis with the conditions described above was
linear (R2=0.994) over the range of 2
to 40 µg of cell lysate, as quantified by densitometry of
autoradiograms analyzed with NIH Image 1.60
software. Densitometry was performed in the linear range of film
development without any processing. All figures were digitized and
processed with Adobe Photoshop and NIH Image to optimize
presentation quality.
Measurement of NO
Ozone chemiluminescent determination of NO oxidation
products (NOx) from BAECs was performed on a Dasibi model
2107 NO analyzer (Glendale, Calif) with a glass reflux chamber
containing 40 mmol/L vanadium III chloride in 3 mol/L HCl. BAECs
were plated onto 6-well tissue culture plates, grown to confluence, and
then treated with cholesterol for 24 hours. Cells were then
washed twice with Krebs-HEPES buffer and incubated for 1 hour in
Krebs-HEPES buffer containing 1 µmol/L A23187. Supernatants were
then collected and assayed for NOx in the chemiluminescent NO
analyzer and quantified with sodium nitrate as a standard.
Protein levels were determined using the Bio-Rad protein assay. Values
are expressed as nmol NOx/mg protein.
Electron Microscopy of BAECs
BAECs were grown in 60-mm dishes until confluent. Cells were
then washed twice in 0.1 mol/L cacodylate buffer (pH 7.4) and
immediately fixed with 2% glutaraldehyde for 1 hour at
room temperature. After fixation, cells were scraped off the dishes,
pelleted, and stored overnight in cacodylate buffer. After serial
dehydration with ethanol and propylene oxide, the cell pellet was
imbedded in LR White (Ted Pella; Redding, Calif), sectioned, and
poststained with uranyl acetate and lead citrate. The electron
microscope grids were visualized and photographed using an electron
microscope (1200ESII; JEOL USA Inc, Peabody, Mass). Only radially
sectioned profiles (20 per treatment) were chosen for analysis.
For quantitation of caveolae, 12 to 15 cells were photographed at a
magnification of x15 000. The photographic negatives were scanned and
imported into Adobe Photoshop. Image analysis included
thresholding and conversion to outlines to generate clearly
identifiable plasma membranes. The image was then imported into NIH
Image 1.60 and membrane length was measured (in µm). Caveolae
were counted by 3 independent blinded observers who used the original
photographs. Data are expressed as mean caveolae number per µm
membrane length.
Oxidation of Caveolae-Enriched Membranes
After isolation of caveolae, 20 µg of caveolae protein was
brought to a final volume of 500 µL with Krebs-HEPES buffer
containing 100 µmol/L xanthine alone (control), 100
µmol/L xanthine plus 0.004 U xanthine oxidase (low oxidation) or
100 µmol/L xanthine plus 0.01 U xanthine oxidase (high
oxidation). Oxidation proceeded for 5 minutes, and then samples were
centrifuged at 100 000g for 1 hour. The
supernatants and pellets were isolated from each sample and placed in a
microcentrifuge tube containing ADP-Sepharose and rotated
overnight at 4°C. Each sample was then washed twice with Krebs-HEPES
buffer, resuspended in Laemmli buffer, and assayed for eNOS by Western
blot.
Statistical Analysis
All results are reported as mean±1 SEM. Western blots were
scanned in the linear range of film development to yield arbitrary
densitometric units. Results were normalized to the control cells in
each experiment to account for variability in cell preparations (3
different BAEC preparations were used). To test for differences in the
relative amounts of proteins present in the subcellular fractions,
normalized densitometric units were analyzed by ANOVA. When
significant F values were obtained, post hoc comparisons were tested
for significance by the Fisher least-significant difference test with
P<0.05 considered to be significant. All statistical tests
were done with SYSTAT software for the Macintosh, version 5.2.
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Results |
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Generation and Measurement of ROS Production
To characterize the effects of ROS on eNOS and caveolin in BAECs, cells were grown to confluence and treated with LY83583. LY83583 is a napthoquinolinedione that generates intracellular superoxide via an NADH-dependent mechanism.29 LY83583 stimulated a concentration- and time-dependent increase in superoxide levels in BAECs, as measured with lucigenin (Figure 1A
). The increase in superoxide
production persisted for at least 1 hour at the plateau level
observed at 20 minutes (not shown). Similar results were obtained with
phenylmethyl sulfonate, another superoxide-generating
compound.27 To verify the production of superoxide
by LY83583, we measured ROS production by oxidation of
aconitase.28 Aconitase is inactivated by
superoxide as a result of oxidation of one Fe(II) in the
[4Fe-4S]–containing active site. The enzyme may be isolated from
cells in the inactive state and enzyme activity determined in vitro.
The ratio of inactive to active enzyme reflects the change in
intracellular redox state.27 28 LY83583 also yielded a
concentration- and time-dependent increase in superoxide formation, as
measured by aconitase oxidation.
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Cholesterol Effects on eNOS and Caveolin-1 Expression
and Subcellular Localization in BAECs
Cholesterol and caveolin are integral components of
caveolae and are required for caveolae formation. Previous studies by
Fielding et al30 showed that treatment of fibroblasts with
cholesterol enhanced caveolin mRNA synthesis. To determine
the effects of cholesterol loading on eNOS and caveolin-1
expression in BAECs, cells were grown to confluence and treated with
free cholesterol for 24 hours. Preliminary data showed that
30 µg/mL cholesterol yielded the greatest increase in
eNOS protein expression, as measured by Western blot analysis.
Similar results were obtained with purified LDL containing equivalent
levels of cholesterol (not shown). Densitometric
analysis of Western blots was used to quantitate eNOS and
caveolin levels. In response to cholesterol, there was a
significant increase in cellular eNOS expression (1.50±0.20, N=6,
P=0.027; Figure 2). The same
Western blots were probed for caveolin, which did not show a
significant increase (1.23±0.34-fold).
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Effect of ROS on BAEC eNOS and Caveolin
To determine the effects of ROS on eNOS and caveolin, we used
1 µmol/L LY83583 to generate superoxide. After 24 hours of
treatment with LY83583, there was a significant decrease in basal eNOS
expression to 0.61±0.08 of control (P=0.05, N=4; Figure 2
).
The addition of LY83583 to cells that were also treated with
30 µg/mL cholesterol prevented the increase in eNOS
observed with cholesterol alone (1.10±0.08-fold relative
to control, N=5, P>0.5 versus control). Because there was a
decrease in eNOS by LY83583 basally, it is not possible to determine
whether there was a positive cholesterol effect in the
presence of LY83583 that "restored" eNOS to control levels.
Nonetheless, LY83583 decreased eNOS expression, both basally and in
response to cholesterol. In contrast, there were no
significant changes in caveolin-1 expression in response to any
treatment (Figure 2). There were also no significant changes in
expression of caveolae-associated proteins dynamin, ERK1/2, or
c-Src.31 The fact that LY83583 inhibited the
cholesterol-mediated increase in eNOS expression, without
change in expression of several other proteins, suggests that ROS did
not cause a toxic effect on the cell by inhibiting protein synthesis,
for example. Thus treatment of BAECs with LY83583 appears to have a
greater effect on expression of eNOS than on expression of several
other cytoplasmic and caveolae-associated proteins.
Effect of Cholesterol and ROS on BAEC Proteins in
Caveolae-Enriched Membranes
To determine whether the subcellular localization of eNOS and
caveolin-1 was altered by cholesterol, we used a
detergent-free method to isolate caveolae-enriched membranes based on
sedimentation gradient centrifugation.21
BAECs were grown to confluence and maintained in 10% FCS or treated
with 30 µg/mL cholesterol. Cells were lysed in hypotonic
buffer, and caveolae-enriched membranes were prepared by OptiPrep
gradient density analysis. This isolation method selectively
enriches proteins associated with caveolae, as shown by the relative
increase in caveolae-associated proteins such as eNOS and caveolin-1
(Figure 3A) as well as dynamin and c-Src
(not shown). Cholesterol treatment of BAECs before caveolae
isolation significantly increased the yield of caveolae-associated
protein by 1.56-fold (P=0.03, N=4). eNOS association with
caveolae-enriched membranes was also increased by
cholesterol treatment relative to control (2.23±0.6-fold,
N=4, P=0.02; Figure 3B). These findings suggest that
cholesterol increased eNOS localization to caveolae.
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Caveolae-enriched membranes were also prepared after treatment with 30
µg/mL cholesterol and 1 µmol/L LY83583 for 24
hours. The addition of LY83583 to BAECs in the presence of
cholesterol decreased the yield of caveolae-associated
protein to 1.02-fold relative to control. LY83583 also decreased the
eNOS association with caveolae-enriched membranes to 1.24-fold relative
to control (Figure 3C). The effect of LY83583 alone was not
investigated, because the results for eNOS and caveolin expression were
clear (Figure 2). Thus, it appeared that LY83583 treatment may
have altered caveolae number or caused dissociation of proteins from
caveolae.
Effect of Cholesterol and ROS on BAEC
Caveolae
To assay qualitatively for caveolae number and structure, caveolae
were visualized by electron microscopy. BAECs were plated on 60-mm
dishes and treated with 30 µg/mL cholesterol±1
µmol/L LY83583 for 24 hours and processed for electron microscopy as
described in Materials and Methods. Caveolae were defined as plasma
membrane–associated vesicles of 50 to 100 nm that were flask-shaped.
Caveolae were prevalent in control BAECs maintained in 10% FCS on both
the luminal and abluminal sides of the cell (Figure 4A). BAECs treated with
cholesterol alone (Figure 4B) displayed
increased numbers of caveolae on both luminal and abluminal sides of
the cell. In BAECs treated with cholesterol and LY83583, a
marked decrease in the total number of caveolae was observed (Figure 4C).
To determine the significance of these changes, caveolae
number were quantified by counting the numbers of caveolae per
µm membrane length. Quantitation revealed significant increases by
cholesterol (relative to control) and a significant
decrease (relative to cholesterol) by
cholesterol plus LY83583 (Table). These findings suggest
that formation and stability of caveolae are altered by ROS.
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Cholesterol Treatment and ROS Alter NO
Production by BAECs
To determine the effect of cholesterol and LY83583
treatment on eNOS activity, NO production was measured by
chemiluminescence assay for NOx. BAECs were grown to confluence and
treated with 30 µg/mL free cholesterol±1 µmol/L
LY83583 for 24 hours. Cells were washed twice and then incubated for 1
hour in Krebs-HEPES buffer containing vehicle or 1 µmol/L
A23187, a calcium ionophore used to stimulate NO production
independent of membrane signal transduction mechanisms. The
supernatants were collected and assayed for NOx as described in
Materials and Methods. In the presence of vehicle, there was no
difference in NOx in response to any of the treatments, indicating that
basal NO production was not altered. In the presence of A23187,
there was a 1.56±0.23-fold increase in NOx from BAECs treated with
free cholesterol compared with control (Figure 5, n=4; P=0.05). This increase
in NOx correlated with the increase in eNOS expression measured by
Western blot (Figure 2). Surprisingly, LY83583 treatment had no
significant effect on NO production in the presence or absence
of cholesterol (Figure 5).
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ROS Dissociate eNOS From Caveolae-Enriched Membranes
To determine whether ROS may directly regulate eNOS association
with caveolae, caveolae-enriched membranes were purified by OptiPrep
gradient density analysis and oxidized in vitro. The
combination of xanthine and xanthine oxidase was used to generate
varying levels of ROS, as measured by superoxide production
(Figure 6A). For these experiments,
LY83583 could not be used because it must be metabolized by NADH
oxidases to generate superoxide. Preliminary studies showed that no
NAD(P)H oxidase or xanthine oxidase activity was present in
caveolae-enriched membranes (not shown). In caveolae-enriched membranes
treated with xanthine alone for 5 minutes (Figure 6B, control),
eNOS remained associated with the particulate fraction after a
100 000g ultracentrifugation for 1 hour
(Figure 6B, ratio of eNOS in supernatant/pellet
0.001, n=3).
Caveolae-enriched membranes exposed to a low level of ROS (100
µmol/L xanthine+0.004 U xanthine oxidase) showed a small increase in
eNOS released from the membranes (ratio of particulate eNOS in
xanthine/xanthine oxidase over control=0.90±0.05). In contrast,
caveolae-enriched membranes exposed to a high level of ROS (100
µmol/L xanthine+0.01 U xanthine oxidase) showed a dramatic increase
in eNOS released from the membranes (ratio of particulate eNOS in
xanthine/xanthine oxidase over control=0.50±0.12, n=3). To determine
the specificity of release of eNOS by ROS, we also measured the release
of caveolin from caveolae (Figure 6C). Caveolin was more
"sensitive" to release by ROS, with low-level ROS stimulating
significant dissociation of caveolin (ratio of particulate caveolin in
xanthine/xanthine oxidase over control=0.46±0.16) at 100 µmol/L
xanthine+0.004 U xanthine oxidase. There was no further caveolin
release by high-level ROS (ratio to control=0.48±0.18). Thus, there
was a close relationship between the magnitude of ROS generation and
dissociation of eNOS from the particulate caveolae fraction. The fact
that eNOS remained associated with caveolae at levels of oxidation that
released 50% of caveolin suggests that eNOS binds to other proteins
in caveolae15 32 33 or there is more than one
population of caveolin molecules, at least 50% of which exist in a
readily releasable state.
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Discussion |
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The major findings of the present study are that ROS inhibit cholesterol-mediated increases in eNOS expression and endothelial cell caveolae number. In addition, we show that ROS dissociate eNOS and caveolin from caveolae-enriched plasma membranes. Despite these complex effects of cholesterol and ROS on eNOS and caveolae, there was no change in NO production induced by calcium ionophore in cultured BAECs. We believe that these results provide insight into the mechanisms by which hypercholesterolemia and oxidative stress in vivo may alter endothelial function. First, it is clear that eNOS expression and caveolae number are increased by cholesterol. These effects would be anticipated to result in increased NO production in response to agonists, which was in fact observed. Second, it is clear that ROS decreased the cholesterol-mediated increases in eNOS and caveolae yet failed to alter NO production. One possible explanation for this finding is the difference in the magnitude of oxidative stress required to dissociate caveolin and eNOS from caveolae membranes. Specifically, although ROS would be predicted to decrease NO production (based on decreased caveolae number and eNOS protein), ROS may paradoxically increase activity of eNOS that remains associated with caveolae (based on decreased binding to the inhibitory protein caveolin and increased [Ca2+]i).34 35 These counterbalancing effects of ROS would represent an endogenous mechanism to compensate for oxidative stress by generating additional NO. Another explanation is that oxidative stress uncouples L-arginine utilization from NO generation or increases formation of peroxynitrite. Both of these mechanisms would not change the amount of NOx measured but would change the amount of active NO that modulates vessel function. In vivo measurements of caveolae number, eNOS expression, and caveolin-1 binding to caveolae under conditions of oxidative stress will be required to determine the physiological importance of our observations.
eNOS function in the endothelium has previously been shown to be regulated rapidly by stimuli that raise calcium (eg, flow and hormones3 ) and chronically by stimuli that modify gene expression (eg, TGF-ß and increased fluid shear stress4 6 ). The present results suggest that ROS and cholesterol are also important regulators of eNOS function. In response to exogenous cholesterol, BAECs expressed higher steady-state levels of eNOS. More importantly, cholesterol stimulated eNOS association with membranes presumed to be caveolae membranes, as determined by OptiPrep density gradient centrifugation. In contrast, ROS decreased steady-state eNOS protein expression, inhibited cholesterol-mediated increases in eNOS, and decreased eNOS association with caveolae. In addition, exposure of caveolae-enriched membranes to xanthine plus xanthine oxidase in vitro caused rapid (5 minutes) dissociation of caveolin and eNOS from the membranes. These results suggest that in vivo, exposure to hypercholesterolemia and ROS will alter both eNOS expression and subcellular localization, which may have important effects on NO production.
The present study is the first to report that ROS alter the expression of eNOS. The effect of ROS appears specific, because there was no change in caveolin-1 expression and no decrease in cell viability. We were unable to study the effect of LY83583 on caveolae number given the relatively small number of caveolae. The finding that there was no increase in caveolin-1 expression in response to cholesterol differs from a previous report.30 Two probable explanations are differences in cell type (BAECs versus fibroblasts) and measurement of protein, as done here, versus measurement of mRNA, as done previously. Of interest, a preliminary report showed that phenolic antioxidants such as nordihydroguaretic acid and vitamin E were able to increase eNOS expression.36 Thus, it appears that cellular redox state may directly regulate eNOS expression.
eNOS is unique among the NOS isoforms in that it is targeted to the plasma membrane, suggesting that subcellular localization is important for eNOS function. The present study demonstrates that both cholesterol and ROS regulate eNOS subcellular localization. By the use of OptiPrep gradient purified membranes, the present study suggests that cholesterol and ROS regulate eNOS subcellular localization by at least 3 mechanisms. (1) Posttranslational modifications of eNOS, including myristoylation and especially palmitoylation, which are required for eNOS association with membranes,11 may be inhibited by ROS as a result of oxidation of critical amino acid residues and inhibition of necessary enzymes. (2) Alterations in protein-protein or protein-lipid interactions such as association of eNOS with caveolin have been suggested to regulate both eNOS localization12 and activity. 37 38 It has recently been shown that caveolin-1 has a scaffolding domain that mediates interactions with multiple caveolae-associated proteins.39 These proteins (including eNOS) contain a conserved motif that appears to be responsible for interacting with the scaffolding domain. It has been proposed that eNOS activation by calcium is inhibited when eNOS is bound to caveolin,37 suggesting that release of eNOS from caveolin is associated with increased NO production. However, the effect on eNOS subcellular localization remains undefined. In addition, Venema et al40 found another eNOS binding protein (termed ENAP-1) that associated with eNOS in response to bradykinin-mediated translocation to the Triton X-100 insoluble fraction. Garcia-Cardena et al15 reported that heat shock protein 90 interacts with eNOS. On the basis of the similarity in molecular weight, it appears likely that ENAP-1 is heat shock protein 90. These data suggest that several proteins present in caveolae may interact with eNOS and regulate eNOS function. The finding that caveolin dissociated from caveolae-enriched membranes at concentrations of ROS lower than required to dissociate eNOS further supports a functional role for other eNOS binding proteins. (3) ROS may disrupt caveolae structural integrity. Increases in membrane cholesterol augmented BAEC caveolae number. Cholesterol has been shown to modify the allosteric properties of eNOS consistent with a change in the fluidity of the lipid microenvironment of the enzyme.41 Whether changes in membrane fluidity affect eNOS subcellular localization remains to be shown definitively. On the other hand, ROS decreased the number of caveolae in BAECs. The decrease in caveolae number likely involves mechanisms that regulate caveolae structural integrity. Of note, a recent study showed that ROS increased tyrosine phosphorylation of caveolin,34 although no functional consequence was reported. In addition, changes in caveolae structural integrity are suggested by the findings that ROS rapidly dissociated caveolin and eNOS from caveolae-enriched membranes and by reports that oxidation of cholesterol may have important consequences for caveolin-1 interactions with the lipid bilayer.24 Thus, the multiple mechanisms that regulate eNOS subcellular location represent potentially novel mechanisms by which cholesterol and ROS modulate eNOS function.
The effects of eNOS localization to plasma membrane and caveolae with regard to eNOS function are not yet fully understood. Several studies have demonstrated that membrane-associated eNOS has greater activity than does cytosolic eNOS.3 8 Data from the present study in general support this mechanism, because we observed that cholesterol increased eNOS localization to membranes and enhanced NO production. However, other studies have shown that eNOS bound to caveolin exhibits decreased NO production due to inhibition of calmodulin binding by caveolin.37 Our data also support this mechanism because treatment with LY83583 caused decreased eNOS association with membranes and caveolae, yet there was little change in calcium ionophore–stimulated NO production. This result may be explained by the fact that ROS-mediated dissociation of caveolin and eNOS from caveolae occurs at different concentrations of ROS, thereby removing a functional inhibitor (caveolin-1) from eNOS. Alternatively, there may be compensatory changes during exposure to ROS that result in greater activity of eNOS.
Decreased NO bioactivity has been proposed as a unifying mechanism for endothelial dysfunction observed in many cardiovascular diseases. The pathogenic importance of endothelial dysfunction in atherosclerosis is supported by the correlation between risk factors for atherosclerosis (hypertension, smoking, and hypercholesterolemia) and development of endothelial dysfunction.42 The nature of endothelial dysfunction in hypercholesterolemia has been well studied, and it has been shown that there is increased NO production43 but decreased bioactivity due to increased destruction (presumably by ROS).44 Alternatively, in humans, it appears that atherosclerosis is associated with a decrease in eNOS expression by endothelial cells that overlay atherosclerotic lesions.45 46 The present study suggests that cholesterol and ROS exert complex effects on eNOS expression and subcellular localization that may result in several effects on eNOS activity. Cultured BAECs exposed to superoxide and cholesterol showed no change in NO production to calcium ionophore. However, in vivo, where cell exposure to hypercholesterolemia occurs in a complex hormonal milieu, the effects on NO production remain unknown. The present study suggests that defining the specific mechanisms by which cholesterol and ROS regulate eNOS subcellular localization and enzyme activity will provide important insights into endothelial dysfunction associated with hypercholesterolemia and atherosclerosis.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL18645 and HL49192) to Dr Bradford C. Berk. These experiments were performed while Dr Berk was an Established Investigator of the American Heart Association. Albert Wu was supported by NIH Medical Scientist Training Grant GM07266, and Chen Yan was supported by NIH training grant T32 HL07828.
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Footnotes |
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This manuscript was sent to Valentin Fuster, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received December 22, 1997; accepted April 20, 1999.
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A. Nuszkowski, R. Grabner, G. Marsche, A. Unbehaun, E. Malle, and R. Heller Hypochlorite-modified Low Density Lipoprotein Inhibits Nitric Oxide Synthesis in Endothelial Cells via an Intracellular Dislocalization of Endothelial Nitric-oxide Synthase J. Biol. Chem., April 20, 2001; 276(17): 14212 - 14221. [Abstract] [Full Text] [PDF]
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J. Hoffmann, J. Haendeler, A. M. Zeiher, and S. Dimmeler TNFalpha and oxLDL Reduce Protein S-Nitrosylation in Endothelial Cells J. Biol. Chem., October 26, 2001; 276(44): 41383 - 41387. [Abstract] [Full Text] [PDF]
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T. J. Guzik, S. Mussa, D. Gastaldi, J. Sadowski, C. Ratnatunga, R. Pillai, and K. M. Channon Mechanisms of Increased Vascular Superoxide Production in Human Diabetes Mellitus: Role of NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase Circulation, April 9, 2002; 105(14): 1656 - 1662. [Abstract] [Full Text] [PDF]
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