 |
INTRODUCTION |
Nitric oxide (NO)1 is
generated in endothelial cells from the conversion of
L-arginine to L-citrulline by the enzymatic
action of an NADPH-dependent NO synthase (NOS) which
requires tetrahydrobiopterin, FAD, and FMN as cofactors (1).
Endothelial NOS (eNOS) is constitutively expressed and activated upon
an increase of intracellular Ca2+ following cell
stimulation with agonists such as thrombin and bradykinin or through
serine phosphorylation subsequent to cell stimulation with shear stress
or insulin (2, 3). Optimal NO formation has been shown to be dependent
on the availability of intracellular cofactors (tetrahydrobiopterin)
(4, 5) and the subcellular localization of the enzyme (6). The eNOS is
primarily membrane-bound and associated with Golgi membranes (7-9) and
plasmalemmal caveolae (10-12) where it is quantitatively associated
with caveolin, the structural coat component of these microdomains. The
complex formation between eNOS and caveolin has been shown to inhibit
enzyme activity, and the inhibitory effect was reversed upon binding of
Ca2+/calmodulin (13-20). The membrane localization of eNOS
is largely dependent on N-myristoylation and cysteine
palmitoylation (cysteine 15 and 26) of the enzyme (7-12, 17, 21).
Interestingly, inhibition of dual acylation or mutation of the
palmitoylation sites leading to an attenuation of Golgi or caveolae
targeting, respectively, were both associated with an impaired cellular
NO synthesis (7, 12).
Endothelium-derived NO exerts vasodilatory, growth regulatory, and
antithrombotic activities thus being an important regulator of
cardiovascular homeostasis (22). Evidence is accumulating that NO
determines the anti-atherosclerotic properties of the endothelium (23).
All major risk factors for atherosclerosis including
hypercholesterolemia, hypertension, and smoking have been associated
with impaired vascular NO synthesis (24). An increasing number of
studies suggest that oxidized/modified low density lipoprotein (LDL),
which is considered to play a key role in the development of
atherosclerosis, may regulate the availability of NO (25). Oxidized LDL
(oxLDL) has been shown to inhibit the NO-mediated
endothelium-dependent vasorelaxation (26-28). The
underlying mechanisms are thought to involve the uncoupling of
Gi protein-dependent signal transduction (29),
an increased inactivation of NO (30-32), and a reduced formation of NO
due to a limited availability of L-arginine (33) or a
decrease in eNOS-mRNA stability and eNOS expression (34, 35).
Recent findings suggest that inhibition of endothelial NO formation by
oxLDL may also be related to an alteration of subcellular eNOS
localization. Blair et al. (36) showed that an acute
treatment of endothelial cells with oxLDL depleted caveolae of
cholesterol and caused eNOS and caveolin-1 to translocate from the
plasma membrane without affecting palmitoylation, myristoylation, or
phosphorylation of the enzyme. Concomitantly, acetylcholine-induced
activation of the enzyme was impaired.
Most studies investigating the effects of oxLDL used
Cu2+-oxidized LDL as an experimental model. In contrast,
there appears to be little evidence that free metal ions play an
important role in the early development of atherosclerosis (25). The
analysis of stable oxidation products in the human artery wall suggests that oxidation of LDL may rather involve the 15-lipoxygenase pathway, reactive nitrogen species, or myeloperoxidase-initiated reactions (25).
Myeloperoxidase is released by activated neutrophils or monocytes and
is catalyzing the reaction of H2O2 with
Cl
, resulting in the formation of hypochlorous acid
(HOCl) (37). This potent oxidant is thought to play an important role
during microbial killing and to contribute to inflammatory tissue
injury and atherogenesis. HOCl has been shown to oxidize LDL in
vitro thereby generating a particle that caused foam cell
formation (38), stimulation of neutrophil adherence to endothelial
cells (39), production of reactive oxygen species by neutrophils (39), interleukin-8 formation by monocytes (40), and an increase in platelet
aggregation (41). Several studies showed the presence of enzymatically
active myeloperoxidase (42), HOCl-oxidized (lipo)proteins (43, 44),
3-chlorotyrosine (45) and dityrosine (46), both markers for
HOCl-derived protein oxidation (47, 48), in human atherosclerotic
lesions supporting the importance of the
myeloperoxidase/H2O2/halide system as a
potential in vivo oxidant. HOCl-modified epitopes have also
been shown to colocalize with myeloperoxidase in lesion material
including endothelial cells (44). Furthermore, HOCl-modified
(lipo)proteins have been detected in inflammatory kidney tissues rich
in myeloperoxidase (49).
In the present study we investigated the effects of
hypochlorite-modified LDL on NO formation, eNOS protein and mRNA
expression as well as subcellular localization of eNOS in human
endothelial cells. We demonstrate that hypochlorite-modified LDL causes
an inhibition of agonist-stimulated NO synthesis without altering the
expression of eNOS, tetrahydrobiopterin availability, or arginine uptake into the cells. The reduced NO formation was associated with a
striking intracellular redistribution of eNOS including the reduction
of the enzyme in the plasma membrane and a disintegration of the
perinuclear Golgi localization. Our data additionally underline the
functional importance of Golgi-targeted eNOS since cyclodextrin that
exclusively caused translocation of plasmalemmal eNOS did not inhibit
cellular NO formation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Plasticware for cell culture was from Greiner
Labortechnik (Frickenhausen, Germany). Medium 199 (M199), human serum
(HS), fetal calf serum, collagenase and human serum albumin (HSA) were from BioWhittaker Europe (Verviers, Belgium).
L-[2,3,4,5-3H]Arginine monohydrochloride (61 Ci/mmol), L-[U-14C]arginine monohydrochloride
(303 mCi/mmol), [9,19-3H]palmitic acid (53 Ci/mmol),
[9,19-3H]myristic acid (54 Ci/mmol),
[32P]dCTP, Megaprime DNA labeling system,
[2-3H]AMP, ammonium salt (21 Ci/mmol),
[3H]cGMP Biotrak radioimmunoassay system, hyperfilm MP,
hyperfilm
max, ECL, hyperfilm ECL,
HybondTM-XL membranes, and PD-10 columns were purchased
from Amersham Pharmacia Biotech. Tran35S-label (>1000
Ci/mmol) and methionine-free RPMI medium were from ICN Biomedical
Research Products (Costa Mesa, CA); En3Hance solution was
purchased from PerkinElmer Life Sciences; nitrocellulose was from
Millipore (Eschborn, Germany); specific antibodies against human eNOS
(monoclonal, clone 3) and human caveolin-1 (monoclonal, clone C060, or
rabbit polyclonal) were obtained from Transduction Laboratories
(Lexington, KY); the monoclonal antibody against giantin was a kind
gift of Dr. Hauri (University of Basel, Switzerland); the alkaline
phosphatase anti-alkaline phosphatase complex (mouse monoclonal) and
the bridging antibody rabbit anti-mouse IgG were purchased from Dako
(Hamburg, Germany); the polyclonal antibody against human eNOS was from
Santa Cruz Biotechnology (Santa Cruz, CA); and the fluorescence-labeled
secondary antibodies (Cy-3- and Cy-2-labeled goat anti-mouse IgG and
goat anti-rabbit IgG, suitable for multilabeling) were obtained from
Jackson ImmunoResearch Laboratories (West Grove, PA). The protease
inhibitor mixture Complete, EDTA-free, and an enzymatic test for the
determination of cholesterol were purchased from Roche Molecular
Biochemicals. Sepiapterin, L-nitroarginine methyl ester
(L-NAME), and the cDNA probe for human eNOS were from
Alexis Corp. (Läufelfingen, Switzerland). Endothelial cell growth
supplement, peroxidase-labeled anti-mouse IgG (Fab-specific),
anti-mouse agarose, FAD, FMN, calmodulin, ionomycin, thrombin, EDTA,
EGTA, trypsin/EDTA solution (0.05/0.02%, v/v), leupeptin,
phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT),
methyl-
-cyclodextrin and other reagents were obtained from Sigma.
The composition of the Hepes buffer (pH 7.4) was as follows: 10 mM Hepes, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 1.5 mM CaCl2. The solubilization buffer contained
100 mM NaOH, 2% Na2CO3, and 1%
SDS. A protease inhibitor mixture stock solution was prepared by
dissolving 1 tablet in 0.5 ml of 100 mM phosphate buffer
(pH 7.0) and stored at
20 °C.
Cell Cultures--
Human umbilical cord vein endothelial cells
(HUVEC) were prepared with 0.05% collagenase and cultured in
75-cm2 plastic flasks in M199 containing 15% fetal calf
serum, 5% HS, and 7.5 µg/ml endothelial cell growth supplement.
Confluent cultures were detached by trypsin/EDTA and plated on glass
coverslips for immunohistochemical stainings, on 30 mm-diameter wells
for the purpose of cGMP determination, on 60 mm-diameter dishes for the measurement of citrulline formation, [14C]arginine
uptake, and incorporation of labeled compounds, and on 90 mm-diameter
dishes for the investigation of the other parameters. Experiments were
carried out with monolayers of the first to second passage.
Isolation and NaOCl Modification of Human LDL--
Plasma from
normolipemic human volunteers was collected in tubes containing 1 mg/ml
EDTA. LDL (d 1.035-1.065 g/ml) was isolated by
ultracentrifugation as described (50). LDL samples were stored under
argon and used within 10 days following isolation. Prior to its
modification LDL was desalted; low molecular mass compounds were
removed by dialysis, and LDL was suspended in phosphate-buffered saline
(PBS). The concentration of the reagent NaOCl was determined spectrophotometrically using a molar absorption coefficient for OCl
of 350 cm
1 at 292 nm. One
mg of LDL protein/ml of PBS was incubated (1 h, 4 °C, under argon)
with NaOCl solution at molar ratios of NaOCl to LDL, ranging from 50 (100 µM NaOCl) to 400 (800 µM NaOCl) with the final pH adjusted to 7.4. The modified LDL preparations were passed
over a PD-10 column to remove unreacted NaOCl. Characterization of
NaOCl-modified LDL revealed that an increasing molar NaOCl:LDL ratio
led to an increased relative electrophoretic mobility and a decreased
percentage of free
-amino groups up to 75% as described previously
(41).
Experimental Incubations--
Preincubation of HUVEC with native
LDL (nLDL) or NaOCl-modified LDL (NaOCl-LDL) suspended in Hepes buffer
(pH 7.4) was performed in culture medium for 1-24 h. The indicated
amounts of LDL used in the experiments were based upon the LDL protein
concentration. If not otherwise announced, NaOCl-LDL modified at a
molar NaOCl:LDL ratio of 400:1 was used. Neither nLDL nor modified LDL
affected endothelial cell viability which was determined by trypan blue exclusion and ranged from 95 to 98% under the different conditions described. None of the tested native or modified lipoproteins altered
the growth behavior of endothelial cells or their capability of protein
synthesis as estimated by incorporation of [3H]thymidine
or 14C-labeled amino acids, respectively. Furthermore, no
evidence of endothelial cell apoptosis was seen when nLDL or NaOCl-LDL were added to culture medium at the indicated concentrations and modifications, which was measured by the cell death detection enzyme-linked immunosorbent assay (Roche Molecular Biochemicals). Sepiapterin was dissolved in dimethyl sulfoxide
(Me2SO). Stimulation of HUVEC with ionomycin or
thrombin was performed in the absence of lipoproteins and sepiapterin.
Ionomycin was dissolved at 1 mM in Me2SO and
stored at
20 °C until used. The final concentration of
Me2SO during experimental incubations and cell stimulation did not exceed 0.1%, and control cells received the same volume addition of solvent.
Immunohistochemical Detection of Hypochlorite-modified LDL in
Endothelial Cells--
Endothelial cells cultured on coverslips were
fixed for 10 min in 1% paraformaldehyde in PBS, washed with PBS, and
permeabilized with methanol. Nonpermeabilized cells were used as a
control. HOCl-modified epitopes were detected with the three-step
alkaline phosphatase anti-alkaline phosphatase method (51). A hybridoma cell line supernatant (clone 2D10G9, 1:10) containing a monoclonal antibody that had been raised against HOCl-modified LDL (50) was used
as a primary antibody. Nuclei were counterstained for 10 min with hematoxylin.
Measurement of Citrulline Synthesis--
Citrulline synthesis
was measured by a modification of a technique described previously
(52). Briefly, cell monolayers were incubated at 37 °C for 30 min in
1.5 ml of Hepes buffer (pH 7.4) containing 0.25% HSA. Subsequently,
cells were stimulated with 2 µM ionomycin or 1 unit/ml
thrombin in the presence of 10 µM L-arginine
and 3.3 µCi/ml L-[3H]arginine. The reaction
was stopped after 15 min with ice-cold PBS containing 5 mM
L-arginine and 4 mM EDTA, and the cells were denaturated with 96% ethanol. After evaporation, the soluble cellular components were dissolved in 20 mM Hepes-Na (pH 5.5) and
applied to 2-ml columns of Dowex AG50WX-8 (Na+ form). The
radioactivity corresponding to the [3H]citrulline content
of the eluate was quantified by liquid scintillation counting.
Agonist-induced [3H]citrulline production was calculated
from the difference in radioactivity from unstimulated and ionomycin-
or thrombin-stimulated cells and was expressed in fmol/mg cell protein.
Basal [3H]citrulline synthesis was determined from the
L-NAME (1 mM, 30 min preincubation)-inhibitable
radioactivity in unstimulated cells and was not always detectable.
Determination of cGMP--
HUVEC monolayers were incubated for
30 min in M199 containing 0.25% HSA and 0.5 mM
isobutylmethylxanthine. Subsequently, the cells were stimulated with 2 µM ionomycin or 1 unit/ml thrombin for 15 min. The
reaction was stopped with 96% ethanol. When the ethanol had
evaporated, 0.3 ml of buffer (50 mM Tris, 4 mM
EDTA (pH 7.5)) were applied. The cGMP content of 100 µl of cellular extract was measured by radioimmunoassay following the instructions of
the manufacturer. The intracellular cGMP concentration was expressed in
pmol/mg cell protein. The agonist-induced cGMP production was
determined from the difference of cGMP content in ionomycin- or
thrombin-stimulated cells and the corresponding unstimulated cells.
[14C]Arginine Uptake into Endothelial
Cells--
HUVEC were incubated in culture medium containing 335 µM L-[14C]arginine (3 mCi/mmol). After various times, incubations were stopped by washing the
cells with ice-cold Hepes buffer (pH 7.4) containing 5 mM
L-arginine. HUVEC were lysed with solubilization buffer; an
aliquot of the lysate was taken to determine the protein content; and
the radioactivity of the remaining sample was measured by liquid
scintillation counting.
Determination of eNOS mRNA Levels by Northern Blot
Analysis--
Total RNA from HUVEC was extracted according to Chirgwin
et al. (53), quantified by UV spectroscopy at 260 nm, and
electrophoretically resolved on 1% agarose, 6% formaldehyde gels (15 µg RNA per lane). RNA was transferred to HybondTM-XL
membranes by vacuum blotting and covalently linked by ultraviolet irradiation (DNA cross-linker, Whatman Biometra, Göttingen,
Germany). Subsequently, blots were stained with methylene blue and
scanned. The 18 S rRNA bands were taken as indicators for equal RNA
loading. Blots were prehybridized and hybridized overnight at 42 °C
with 2 × 106 cpm/ml of a
[32P]dCTP-labeled fragment of human eNOS-cDNA
according to standard protocols. Radioactivity was visualized by
autoradiography at
80 °C for 20 h.
Preparation of Cell Lysates and Particulate Fractions for Western
Blotting Analysis--
HUVEC were detached by trypsin/EDTA and
suspended in a small volume of Hepes/sorbitol buffer (10 mM
Hepes, 340 mM sorbitol, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.6 mM
leupeptin, and 0.025 mM pepstatin (pH 7.4)). For
preparation of a particulate and a cytosolic fraction, cell suspensions
were sonicated on ice and centrifuged for 1 h at 100,000 × g and 4 °C. Proteins were solubilized by boiling the
cells as well as the soluble and particulate fractions in Laemmli
sample buffer.
SDS Electrophoresis and Immunoblotting--
The samples were
separated by SDS-PAGE on 7% gels (cell lysates, particulate, and
cytosolic fractions) or on 5-15% gradient gels (plasma
membrane-enriched fractions, immunoprecipitates) and blotted onto
nitrocellulose membranes. Subsequently, blots were subjected to
immunostaining with monoclonal antibodies against human eNOS (1:250,
1.5 h) or against human caveolin-1 (1:250, 1.5 h). After an
incubation with a peroxidase-conjugated secondary antibody (1:1000,
1.5 h), visualization of NOS or caveolin-1 was achieved using the
ECL technique.
Immunofluorescence Microscopy--
HUVEC cultured on glass
coverslips were fixed for 10 min at 4 °C with 1% paraformaldehyde
in PBS. Subsequently, cells were washed with PBS and incubated with 5%
goat serum in PBS containing 0.06% saponin for permeabilization. Cells
were then incubated for 30 min with primary antibodies against eNOS
(mouse monoclonal 1:200 or rabbit polyclonal 1:200), caveolin-1 (rabbit
polyclonal, 1:200), and the Golgi marker protein giantin (mouse
monoclonal, 1:1500) which was followed by a 30-min incubation with
Cy-2- or Cy-3-labeled secondary antibodies (1:100 or 1:600,
respectively). Controls with nonimmune IgG and secondary antibody alone
were negative. For colocalization experiments the following antibodies were combined: monoclonal anti-eNOS/polyclonal anti-caveolin-1 and
Cy-3-labeled anti-mouse IgG/Cy-2-labeled anti-rabbit IgG; polyclonal
anti-eNOS/monoclonal anti-giantin and Cy-3-labeled anti-rabbit
IgG/Cy-2-labeled anti-mouse IgG. Cells were observed with a laser
scanning confocal microscope (Zeiss LSM 510, Jena, Germany).
Sucrose Gradient Fractionation and Characterization of a Plasma
Membrane-enriched Subcellular Fraction--
HUVEC were scraped into
ice-cold Hepes/sucrose buffer (10 mM Hepes (pH 7.8), 250 mM sucrose, 1 mM EDTA containing 10 µl stock solution of protease inhibitor mixture/ml) and sedimented by
centrifugation at 1400 × g for 10 min. The pellet was
resuspended in 1 ml of the same buffer and homogenized 20 times with a
tight Dounce homogenizer. The homogenate was layered on top of 12 ml of
a 1-3 M linear sucrose gradient in 10 mM Hepes
(pH 7.8) and centrifuged for 1.5 h at 40,000 × g
and 4 °C in a Beckman SW-40 Ti swinging bucket rotor. 1-ml fractions
were then collected from the top of the gradient, and fractions 2 and 3 containing cellular membranes were pelleted by centrifugation
(100,000 × g, 1 h, 4 °C). The membranes were resuspended in buffer (20 mM Hepes, 10 mM
potassium oxalate, 100 mM KCl, 10 mM
MgCl2 (pH 7.6)) and layered over 3.5 ml of 40% sucrose solution in PBS as described by Enouf et al. (54). After
centrifugation for 1.5 h at 95,000 × g and
4 °C in a swinging bucket rotor (Beckman SW-60 Ti), a band at the
sample sucrose interface was recovered. This fraction was centrifuged
for 1 h at 100,000 × g and 4 °C, resuspended
in Hepes/sucrose buffer, and characterized as plasma membrane-enriched
fraction by an increase of cholesterol content (566 ± 13 versus 329 ± 20 nmol/mg membrane protein in
mixed membrane fractions) and of 5'-nucleotidase activity (62 ± 5.8 versus 31 ± 3.7 nmol/min/mg membrane protein in
mixed membrane fractions). Cholesterol was measured according to the
cholesterol oxidase:p-aminophenazone method using a test kit
provided by Roche Molecular Biochemicals. The activity of the plasma
membrane marker enzyme 5'-nucleotidase was determined according to
Avruch and Wallach (55). Aliquots of the plasma membrane-enriched
fraction were solubilized in Laemmli buffer and subjected to SDS-PAGE
and immunoblotting as described above.
Coimmunoprecipitation--
HUVEC monolayers were lysed with
octyl glucoside buffer (50 mM Tris, 125 mM NaCl
(pH 7.4), containing 60 mM octyl glucoside, 2 mM DTT, 0.05 mM EDTA, 1 mM PMSF,
0.6 mM leupeptin, and 0.025 mM pepstatin).
Immunoprecipitation was performed with a monoclonal anti-eNOS antibody
(8 µg per 500 µg of lysate protein, 4 °C, 2 h) that was
followed by a 1-h incubation with anti-mouse agarose. Precipitated
proteins were washed three times with octyl glucoside buffer and once
with 50 mM Tris, 150 mM NaCl (pH 7.4), eluted in Laemmli sample buffer and subjected to SDS-PAGE and immunoblotting. Blots were stained against caveolin-1 first, then washed in 20 mM Tris, 500 mM NaCl, 0.05% Tween and
counterstained against eNOS as described.
Metabolic Labeling and Immunoprecipitation of eNOS--
HUVEC
were incubated with nLDL or NaOCl-LDL in culture medium for 12 h.
Subsequently, 50 µCi of Tran35S-label reagent in
methionine-free RPMI medium containing the lipoprotein preparations was
added for 3 h. In labeling experiments with fatty acids the 12-h
treatment with lipoproteins was followed by a 30-min incubation in M199
containing nLDL or NaOCl-LDL, 0.25% fatty acid free bovine serum
albumin, and 2 µg/ml cerulenin, a fatty acid synthesis inhibitor.
Cells were then labeled with [3H]palmitic acid or
[3H]myristic acid (300 µCi/ml) in the same medium for 4 or 8 h, respectively. The 3H-fatty acids were dried
under N2 and redissolved in Me2SO so that the
final concentration of the solvent in the labeling medium was 0.1%.
Untreated control cells were processed in parallel. The labeled cells
were lysed for 15 min at 4 °C by the addition of Hepes buffer (pH
7.4) containing 0.5% Triton X-100, 0.05% SDS, and protease inhibitors
(1 mM PMSF, 0.6 mM leupeptin, 0.025 mM pepstatin). Immunoprecipitation was performed with
anti-eNOS antibody as described above. The precipitates were washed
with PBS containing 0.1% Triton X-100, 0.1% SDS, and 0.2% HSA and
eluted by boiling in Laemmli sample buffer (without the addition of
reducing agent to [3H]palmitic acid-labeled samples).
Immunoprecipitated proteins were analyzed by SDS-PAGE and
immunoblotting/autoradiography (35S) or fluorography
(3H). The autoradiography of Western blots was performed
with hyperfilm
max at
20 °C for 2-10 days. For
fluorography gels were treated with En3Hance as directed by
the manufacturer and exposed to hyperfilm MP for 3 weeks.
Protein Determination--
LDL proteins, cell proteins lysed
with solubilization buffer and proteins in cell homogenates and
membrane fractions were measured according to Lowry using the Bio-Rad
DC protein microassay with bovine serum albumin as standard. Protein
determination in cell homogenates containing DTT was performed by the
Bradford method applying the same standard.
Statistical Analysis--
Each experimental point was performed
in duplicate. All data are given as means ± S.E. of three to six
independent experiments. To determine the statistical significance of
the described results, analysis of variance with Bonferroni's
correction for multiple comparisons or Student's t test for
unpaired data was performed. A p value of <0.05 was
accepted as statistically significant.
 |
RESULTS |
Uptake of Hypochlorite-modified LDL into Endothelial
Cells--
HUVEC preincubated with 200 µg/ml NaOCl-LDL for 24 h
in culture medium and labeled with a monoclonal antibody against
HOCl-modified epitopes under permeabilizing conditions showed a strong
granular staining throughout the cytoplasm (Fig.
1A). Since staining was not
seen in nonpermeabilized cells preincubated with NaOCl-LDL (Fig.
1B), these data indicate an intracellular uptake of modified lipoproteins. HUVEC incubated with nLDL (200 µg/ml, 24 h) did not express HOCl-modified epitopes (Fig. 1, C and
D).

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Fig. 1.
Uptake of hypochlorite-modified LDL by
endothelial cells. HUVEC cultured on coverslips were preincubated
with 200 µg/ml NaOCl-LDL (A and B) or nLDL
(C and D) for 24 h in culture medium.
Subsequently, cells were washed, fixed, and labeled with
anti-HOCl-modified LDL IgG (clone 2D10G9) under permeabilizing
(A and C) or nonpermeabilizing (B and
D) conditions. Nuclei were counterstained with hematoxylin.
One representative experiment out of three is shown.
|
|
Effect of Hypochlorite-modified LDL on Citrulline and cGMP
Formation--
NO production upon endothelial cell stimulation is
accompanied by an increased synthesis of citrulline that is produced
stoichiometrically with NO, and by an accumulation of intracellular
cGMP that is generated when NO activates the soluble guanylate cyclase
of the cells. Accordingly, both parameters indicate the formation of NO. Stimulation of HUVEC with ionomycin led to a
[3H]citrulline formation of 249 ± 37 fmol/mg
cellular protein (n = 6) and a cGMP accumulation of
7.0 ± 0.9 pmol/mg protein (n = 4) (Fig.
2A). The response upon
thrombin stimulation was generally lower (80 ± 15 fmol of
[3H]citrulline/mg protein (n = 6) and
2.9 ± 0.1 pmol of cGMP/mg of protein (n = 4))
(Fig. 2B). Pretreatment of HUVEC with nLDL (50-200 µg/ml,
24 h) had no effect on agonist-induced citrulline or cGMP
synthesis (Fig. 2, A and B). NaOCl-LDL (50-200
µg/ml, 24-h preincubation with cells) inhibited citrulline production in comparison to nLDL in a concentration-dependent manner
up to 39 (ionomycin-stimulation) or 73% (thrombin-stimulation) (Fig. 2, A and B). Similarly, agonist-induced cGMP
formation was decreased in HUVEC pretreated with NaOCl-modified LDL (up
to 51 and 59% inhibition of ionomycin- and thrombin-stimulated cGMP
accumulation, respectively, compared with control values in
nLDL-treated cells) (Fig. 2, A and B). Neither
nLDL nor NaOCl-LDL affected basal synthesis of
[3H]citrulline and cGMP. Both agonist-induced citrulline
and cGMP production in untreated HUVEC and in cells preincubated with
nLDL or NaOCl-LDL were entirely blocked by a 30-min preincubation with 1 mM of the NOS inhibitor L-NAME (data not
shown). The inhibitory effect of NaOCl-LDL on ionomycin-stimulated
citrulline formation was time-dependent. After 6, 12, and
24 h preincubation of HUVEC with 200 µg/ml NaOCl-LDL, inhibition
compared with nLDL-treated cells was 7, 15, and 42%, respectively
(Fig. 3). A 1-h treatment of cells with
NaOCl-LDL was not effective in inhibiting calcium-dependent citrulline formation.

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Fig. 2.
Effect of hypochlorite-modified LDL on
citrulline and cGMP formation in endothelial cells. HUVEC were
incubated with 50-200 µg/ml nLDL or NaOCl-LDL for 24 h in
culture medium. Subsequently, the cells were stimulated in Hepes buffer
(pH 7.4) for 15 min with 2 µM ionomycin (A) or
1 unit/ml thrombin (B) and processed for either citrulline
or cGMP measurement. Results (mean ± S.E., n = 5)
are shown as agonist-induced [3H]citrulline formation or
cGMP production calculated from the differences between stimulated and
unstimulated cells and expressed in fmol/mg or pmol/mg cell protein,
respectively. Cells preincubated with equal amounts of nLDL or
NaOCl-LDL were compared, *p < 0.05.
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Fig. 3.
Time dependence of the effect of
hypochlorite-modified LDL on ionomycin-induced citrulline
formation. HUVEC were incubated with 200 µg/ml nLDL or NaOCl-LDL
in culture medium for the indicated times. Cells were then stimulated
in Hepes buffer (pH 7.4) with 2 µM ionomycin in the
presence of 3.3 µCi/ml L-[3H]arginine for
15 min. The generated [3H]citrulline was separated from
[3H]arginine by cation exchange chromatography and
measured by liquid scintillation counting. Data are shown as
agonist-induced increase in [3H]citrulline production
calculated from the differences between stimulated and unstimulated
cells (mean ± S.E., n = 2).
|
|
Generally, the experiments were performed with NaOCl-LDL modified at a
molar NaOCl:LDL ratio of 400:1. LDL modified at lower molar NaOCl:LDL
ratios, however, had similar inhibitory effects on citrulline
formation. Preincubation of HUVEC for 24 h with 200 µg/ml
NaOCl-LDL modified at NaOCl:LDL ratios of 50:1, 100:1, 200:1, and 400:1
caused a ionomycin-triggered [3H]citrulline production of
150 ± 24, 153 ± 7, 145 ± 4, and 130 ± 8 fmol/mg, respectively, whereas 220 ± 23 fmol/mg was measured in
control cells treated with nLDL under identical conditions (n = 3).
Effect of Sepiapterin on NaOCl-LDL-induced Inhibition of Citrulline
Formation--
To investigate whether the inhibitory effect of
NaOCl-LDL on eNOS activity could be overcome by increasing the
concentration of its cofactor tetrahydrobiopterin, incubations of HUVEC
with lipoproteins were performed in the presence of sepiapterin which is intracellularly converted into tetrahydrobiopterin via a salvage pathway (56). Pretreatment of HUVEC with sepiapterin (10 µM, 24 h) led to a 2.5-fold increase in
ionomycin-triggered citrulline formation that was not altered when nLDL
was added in parallel (200 µg/ml, 24 h) (Table I
). When HUVEC were coincubated with sepiapterin and NaOCl-LDL (200 µg/ml, 24 h), a similar
inhibition of ionomycin-induced citrulline synthesis compared with
nLDL-treated HUVEC as seen in cells without the addition of sepiapterin
was observed (52 versus 51%, respectively) (Table I).
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Table I
Effect of sepiapterin on NaOCl-LDL-induced inhibition of citrulline
formation
HUVEC were preincubated for 24 h with 200 µg/ml nLDL or
NaOCl-LDL in culture medium in the absence or presence of 10 µM sepiapterin. Subsequently, the cells were stimulated
in Hepes buffer (pH 7.4) for 15 min with 2 µM ionomycin
in the presence of 3.3 µCi/ml L-[3H]arginine.
The generated [3H]citrulline was separated from
[3H]arginine by cation exchange chromatography and measured
by liquid scintillation counting. Data are shown as agonist-induced
[3H]citrulline formation calculated from the differences in
radioactivity from stimulated and unstimulated cells and expressed in
fmol/mg cell protein (mean ± S.E., n = 3); nLDL-
and NaOCl-treated cells were compared.
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Effect of Hypochlorite-modified LDL on Arginine Uptake into
Endothelial Cells--
To determine whether NaOCl-modified LDL affects
the transport of the NOS substrate arginine into the cells, HUVEC were
incubated for 1-24 h in culture medium containing 335 µM
L-[14C]arginine (3 mCi/mmol) in the presence
of 200 µg/ml nLDL or NaOCl-LDL. Untreated control cells were
incubated in parallel. Table II shows that the [14C]arginine uptake was
time-dependent and was not altered by lipoproteins.
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Table II
Effect of hypochlorite-modified LDL on arginine uptake into endothelial
cells
HUVEC were incubated in culture medium containing 335 µM
L-[14C]arginine (3 mCi/mmol) in the presence or
absence of 200 µg/ml nLDL or NaOCl-LDL for the indicated times. The
radioactivity of cell lysates was measured by liquid scintillation
counting. Control values were obtained by adding and immediately
removing the [14C]arginine medium. Data are shown as cpm of
incorporated [14C]arginine/mg cell protein (mean ± S.E., n = 3).
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Effect of Hypochlorite-modified LDL on eNOS Expression and de Novo
Synthesis--
Northern blot analysis of RNA extracted from untreated
endothelial cells and cells preincubated for 24 h with 200 µg/ml
nLDL or NaOCl-LDL showed no difference in eNOS mRNA expression
under the conditions examined (Fig.
4A). Accordingly,
immunoprecipitation experiments comparing eNOS protein in
35S-labeled control HUVEC and cells pretreated with nLDL or
NaOCl-LDL (200 µg/ml, 12-h preincubation and 3-h coincubation with
Tran35S-label reagent) showed that the
35S-labeled eNOS band was not different between control and
lipoprotein-pretreated cells, suggesting that eNOS de novo
synthesis was not altered by nLDL or NaOCl-LDL (Fig. 4B). A
comparable eNOS protein expression in cell lysates and subcellular
fractions from untreated endothelial cells and cells incubated for
24 h with 200 µg/ml nLDL or NaOCl-LDL was confirmed in Western
blotting studies. Fig. 4C shows that the eNOS protein was
mainly located in the particulate fraction. Differences in eNOS
expression were not seen in whole lysates or in particulate or
cytosolic fractions between untreated and lipoprotein-treated
cells.

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Fig. 4.
eNOS expression and de novo
synthesis in endothelial cells treated with hypochlorite-modified
LDL. HUVEC were incubated with 200 µg/ml nLDL or NaOCl-LDL in
culture medium for 24 h (A and C) or for
12 h followed by 3 h of coincubation with
Tran35S-label reagent (B). A, RNA was
harvested, separated by electrophoresis on 1% agarose, 6%
formaldehyde gels (15 µg/lane), blotted on HybondTM-XL
membranes, and hybridized overnight with
[32P]dCTP-labeled probes for human endothelial NO
synthase (eNOS). rRNA stained with methylene blue served as
a loading control. B, equal amounts of lysates of
35S-labeled cells were immunoprecipitated with a monoclonal
antibody against human eNOS. Immunoprecipitates were separated by
SDS-PAGE on 7% gels, blotted onto nitrocellulose membranes, and
subjected for 10 days to autoradiography and subsequent immunostaining.
C, intact cells as well as particulate and cytosolic
fractions were solubilized with Laemmli buffer. SDS-PAGE was performed
on 7% gels (50 µg/lane), and immunostaining was accomplished with a
monoclonal antibody against eNOS. Representative figures from each
experiment performed three times with similar results are shown.
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Effect of Hypochlorite-modified LDL on Localization of
eNOS--
Localization of eNOS in control cells by indirect
immunofluorescence revealed a strong crescent-shaped perinuclear
staining, a faint fluorescence pattern diffusely distributed throughout the cell, and a discrete staining in the plasma membrane. Confocal microscopy showed a colocalization of eNOS with giantin, an integral protein of the Golgi membranes (57), and partially with caveolin-1 which was primarily localized at the leading edge and in the
perinuclear region (Fig. 5, A
and B). Pretreatment of HUVEC with nLDL (200 µg/ml,
24 h) did not affect eNOS distribution nor its colocalization with
giantin and caveolin-1, respectively (Fig. 5, A and
B). However, a marked intracellular redistribution of eNOS
was observed after preincubating HUVEC with NaOCl-LDL (200 µg/ml,
24 h). Plasmalemmal staining of eNOS was considerably decreased,
and the distinct granular staining in the perinuclear region was
changed into a diffuse cytoplasmic fluorescence pattern. Additionally,
colocalization with giantin or caveolin-1 was abolished or largely
diminished, respectively.

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Fig. 5.
Effect of hypochlorite-modified LDL on the
localization of eNOS in endothelial cells. HUVEC cultured on
coverslips were incubated with 200 µg/ml nLDL or NaOCl-LDL in culture
medium for 24 h. Subsequently, cells were washed, fixed, and
labeled with the following antibodies. A, polyclonal
anti-eNOS and monoclonal anti-giantin followed by Cy-3-labeled
anti-rabbit IgG and Cy-2-labeled anti-mouse IgG. B,
monoclonal anti-eNOS and polyclonal anti-caveolin-1 followed by
Cy-3-labeled anti-mouse IgG and Cy-2-labeled anti-rabbit IgG. Cells
were observed with a laser scanning confocal microscope (Zeiss LSM
510). The bar represents 20 µm. One representative
experiment out of five is shown.
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Effect of Hypochlorite-modified LDL on the Association of eNOS and
Caveolin-1 with a Plasma Membrane-enriched Subcellular
Fraction--
The NaOCl-LDL-induced mislocalization of eNOS was
confirmed by Western blot experiments investigating a plasma
membrane-enriched subcellular fraction prepared from control and
lipoprotein-pretreated cells. Preincubation of HUVEC with NaOCl-LDL
(200 µg/ml, 24 h) led to a reduction of eNOS in this membrane
fraction, whereas no differences in eNOS expression were seen between
untreated and nLDL (200 µg/ml, 24 h)-treated cells (Fig.
6). In contrast, caveolin-1 was similarly
expressed in plasma membrane-enriched fractions from control and
lipoprotein-pretreated cells (Fig. 6).

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Fig. 6.
Effect of hypochlorite-modified LDL on the
association of eNOS and caveolin-1 with a plasma membrane-enriched
subcellular fraction. After preincubation of HUVEC with 200 µg/ml nLDL or NaOCl-LDL in culture medium for 24 h, cells were
scraped, homogenized, and fractionated using a linear 1-3
M sucrose gradient. The mixed membrane fraction was
recovered and centrifuged on a 40% sucrose cushion to obtain a plasma
membrane-enriched subcellular fraction. Aliquots of these fractions
were eluted in Laemmli sample buffer, separated by 5-15% SDS-PAGE
gradient gels (20 µg of protein per lane), and blotted onto
nitrocellulose membranes. Immunostaining was performed with primary
monoclonal antibodies against human eNOS or human caveolin-1
(cav-1). The blot is a representative of two independent
experiments.
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Effect of Hypochlorite-modified LDL on Coimmunoprecipitation of
eNOS and Caveolin-1--
Since NaOCl-LDL treatment of cells affected
the translocation of eNOS but not of caveolin-1 from plasma
membrane-enriched subcellular fractions, we tested whether the observed
changes in eNOS localization were associated with alterations in
eNOS-caveolin interactions. Fig. 7 shows
that monoclonal antibodies against eNOS immunoprecipitated equal
amounts of eNOS from lysates of control and lipoprotein-treated cells.
However, compared with control or nLDL-treated cells, eNOS antibodies
coimmunoprecipitated significantly less caveolin-1 from lysates of
HUVEC preincubated with NaOCl-LDL (200 µg/ml, 24 h) (Fig. 7).
The total caveolin-1 expression in control cells and
lipoprotein-treated cells was identical (data not shown).

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Fig. 7.
Effect of hypochlorite-modified LDL on
interactions of eNOS with caveolin-1. HUVEC were incubated with
200 µg/ml nLDL or NaOCl-LDL in culture medium for 24 h.
Subsequently, cells were lysed, and equal amounts of lysates were
immunoprecipitated with a monoclonal antibody against human eNOS.
Precipitated proteins were eluted in Laemmli sample buffer, separated
by 5-15% SDS-PAGE gradient gels, and blotted onto nitrocellulose
membranes. Immunostaining was performed with a primary monoclonal
antibody against human caveolin-1 (cav-1, lower panel).
Thereafter, blots were washed and counterstained with a monoclonal
anti-eNOS antibody (upper panel). A typical experiment out
of three is shown. IP, immunoprecipitation; IB,
immunoblot.
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Effect of Hypochlorite-modified LDL on Acylation of
eNOS--
Because of the importance of fatty acylation for the
intracellular compartmentalization of eNOS (7-12, 17, 21), we
investigated whether the NaOCl-LDL-induced mislocalization of eNOS was
related to an alteration of myristoylation or palmitoylation of the
enzyme. HUVEC preincubated for 24 h with 200 µg/ml nLDL or
NaOCl-LDL were radiolabeled with [3H]palmitate or
[3H]myristate in the presence of lipoproteins. Fig.
8 shows that the steady-state
palmitoylation or myristoylation of eNOS was not affected by treatment
with either nLDL or NaOCl-LDL.

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Fig. 8.
Myristoylation and palmitoylation of eNOS in
endothelial cells treated with hypochlorite-modified LDL. HUVEC
were incubated with 200 µg/ml nLDL or NaOCl-LDL in culture medium for
12 h. Cells were then labeled in the presence of lipoproteins with
[3H]palmitic acid (A) or
[3H]myristic acid (B) (300 µCi/ml) for 4 or
8 h, respectively. Equal amounts of lysates of labeled cells were
immunoprecipitated with a monoclonal antibody against human eNOS.
Immunoprecipitates were separated by 7% SDS-PAGE gels, and subjected
to fluorography. Representative data from one out of three independent
experiments are shown.
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Effect of Hypochlorite-modified LDL on the Cholesterol Content of
Mixed Membranes and Plasma Membrane-enriched Subcellular
Fractions--
We next analyzed whether the NaOCl-LDL-induced
translocation of eNOS was related to changes in the cholesterol content
of cellular membranes since plasma membrane and caveolar localization of eNOS have been shown to depend on membrane cholesterol (36, 58).
Cholesterol was measured in mixed membrane fractions and plasma
membrane-enriched subcellular fractions prepared from control cells and
cells preincubated for 24 h with 200 µg/ml nLDL or NaOCl-LDL. As
shown in Table III no differences in
membrane cholesterol content were seen between control and
lipoprotein-treated cells.
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Table III
Effect of hypochlorite-modified LDL on cholesterol content of
cellular membranes
After preincubation of HUVEC with 200 µg/ml nLDL or NaOCl-LDL in
culture medium for 24 h, cells were scraped and homogenized. The
mixed membrane fraction was prepared from the cell homogenate by
centrifugation on a linear 1-3 M sucrose gradient, and the
plasma membrane-enriched subcellular fraction was recovered after
centrifugation of the mixed membranes on a 40% sucrose cushion.
Cholesterol was determined in aliquots of membrane suspensions
according to the cholesterol oxidase: p-aminophenazone
method with a test kit provided by Roche Molecular Biochemicals. Data
are shown as nmol of cholesterol/mg of membrane protein (mean ± S.E., n = 3).
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Effect of Cyclodextrin on the Localization of eNOS and the
Formation of Citrulline and cGMP--
To compare the NaOCl-induced
translocation of eNOS with a redistribution of eNOS that is caused by
cholesterol depletion of plasma membranes, we treated cells with
cyclodextrin, a compound known to extract cholesterol from caveolae
(59). Fig. 9 shows that the addition of
cyclodextrin (5 mM, 1 h) to the cells led to a
disappearance of eNOS and caveolin-1 from the plasma membrane. However,
in contrast to the changes seen after treatment of cells with
NaOCl-LDL, the perinuclear localization of eNOS and its colocalization with caveolin-1 and giantin in the Golgi area were maintained.

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Fig. 9.
Effect of cyclodextrin on the localization of
eNOS in endothelial cells. HUVEC on coverslips were incubated with
5 mM cyclodextrin in culture medium for 1 h.
Subsequently, cells were washed, fixed, and labeled with the following
antibodies. A, polyclonal anti-eNOS and monoclonal
anti-giantin followed by Cy-3-labeled anti-rabbit IgG and Cy-2-labeled
anti-mouse IgG. B, monoclonal anti-eNOS and polyclonal
anti-caveolin-1 followed by Cy-3-labeled anti-mouse IgG and
Cy-2-labeled anti-rabbit IgG. Cells were observed with a laser scanning
confocal microscope (Zeiss LSM 510). The bar represents 20 µm. A representative experiments out of five is shown.
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Preincubation of HUVEC with cyclodextrin (5 mM, 1 h)
led to an increase of ionomycin-stimulated [3H]citrulline
formation from 188 ± 42 fmol/mg protein in untreated cells to
256 ± 47 fmol/mg (n = 3, not significant).
Correspondingly, ionomycin-triggered cGMP formation was enhanced from
6.6 ± 0.5 pmol/mg protein to 9.2 ± 1.2 pmol/mg
(n = 3, not significant). Similar results were
obtained when cells were stimulated with thrombin which led to a
[3H]citrulline generation of 99 ± 20 and 144 ± 25 fmol/mg in untreated and cyclodextrin-treated cells,
respectively, and to a cGMP formation of 3.0 ± 0.7 pmol/mg
(control cells) and 3.8 ± 0.9 pmol/mg (cyclodextrin-treated cells) (n = 3, not significant).
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DISCUSSION |
The present study demonstrates that hypochlorite-modified LDL
inhibits agonist-induced endothelial NO synthesis in a dose- and
time-dependent manner. This was shown by a concomitant
decrease of both the formation of citrulline, which is produced
stoichiometrically with NO, and the accumulation of intracellular cGMP,
which is generated upon NO-mediated activation of the soluble guanylate cyclase.
The effect of NaOCl-LDL on NO synthesis is likely to be related to an
uptake of the particles into the cells since cell stimulation was
performed in the absence of extracellular lipoproteins. Indeed, immunohistochemical findings of the present study show the occurrence of HOCl-modified epitopes in cells that had been incubated with NaOCl-LDL, and binding and internalization of hypochlorite-modified lipoproteins might have been mediated by scavenger receptors present on
endothelial cells (58, 60-62). Hypochlorite-modification of LDL has
been shown to result in an immediate oxidation of amino acid residues
with lysine, tryptophan, cysteine, methionine, and tyrosine being the
major targets (38, 41, 63). Some of the initial reaction products
identified as protein-bound chloramines undergo homolytic reactions to
give radicals that initiate LDL lipid oxidation with the generation of
hydroperoxides and hydroxides of cholesteryl esters (64). The latter
might be likely candidates to mediate the inhibiting effects of
hypochlorite-modified LDL on NO formation and may also account for the
kinetics of this effect since they have been shown to accumulate
intracellularly (65). Interestingly, cholesterol oxides have already
been demonstrated to impair endothelial-dependent arterial
relaxation and NO synthesis in HUVEC (66, 67). At present, however, we
have no evidence whether the lipid or the protein moiety of NaOCl-LDL
or both are contributing to the impairment of NO synthesis observed in
our study.
Several inhibitory mechanisms of oxLDL on NO synthesis reported in the
literature (29, 33-35) could be excluded as responsible for the
effects observed with hypochlorite-modified LDL in the present study.
Our findings suggest that NaOCl-LDL acts downstream from signal
transduction required for thrombin-induced NOS activation since NO
formation was also reduced when NaOCl-LDL-treated cells were challenged
with the calcium ionophore ionomycin. The effect of
hypochlorite-modified LDL was not due to a deficiency of the NOS
cofactor tetrahydrobiopterin since the inhibition of NO synthesis was
measured even when the intracellular tetrahydrobiopterin levels were
increased by coincubation of cells with sepiapterin (56). Likewise, a
deficiency of the NOS substrate L-arginine did not account
for the observed effects because NaOCl-LDL did not alter the cellular
uptake of this amino acid. Hypochlorite-modified LDL also did not
affect de novo synthesis of NOS as measured in 35S-labeled cells. Accordingly, neither the amount of
specific mRNA nor the expression of eNOS protein recognized with
specific monoclonal antibodies in cell lysates were altered by
pretreatment of cells with NaOCl-LDL. However, our findings suggest
that the mechanism underlying the inhibition of agonist-stimulated NO
formation by hypochlorite-modified LDL involves an alteration of the
subcellular localization of eNOS. Immunofluorescence studies showed a
distinct eNOS localization in the plasma membrane and in the Golgi
region in control cells and nLDL-treated cells. In contrast, in
endothelial cells preincubated with NaOCl-LDL eNOS was translocated
from both compartments and revealed a diffuse cytoplasmic distribution. However, NaOCl-LDL did not alter the structure of the Golgi region as
characterized by the Golgi marker protein giantin (57) and did also not
lead to major changes of the distribution of caveolin-1 which is known
to be colocalized in part with eNOS (9, 10). The results of the
immunofluorescence microscopy were confirmed by subcellular
fractionation data. Hypochlorite-modified LDL led to a reduction of
eNOS association with a plasma membrane-enriched subcellular fraction,
whereas caveolin-1 expression in this fraction was maintained.
Interestingly, the overall membrane association of eNOS as shown by
immunoblotting analysis of 100,000 × g membrane fractions was not modified by NaOCl-LDL. These findings confirm previous studies that demonstrated that a stable membrane association is not sufficient for proper membrane targeting (8) and further suggest
a translocation of eNOS to a membrane compartment distant from plasma
or Golgi membranes.
Our results are in line with previous studies demonstrating that NOS
mislocalization is paralleled by an attenuated capacity for NO
production in intact cells (7, 12). Different studies using cells
transfected with myristoylation-deficient and/or
palmitoylation-deficient eNOS mutants revealed that targeting of eNOS
to caveolae or Golgi membranes is regulated in an
acylation-dependent manner (7-12, 17, 21). In our
experimental approach, however, translocation of eNOS from plasma
membrane and Golgi was not associated with changes in the steady-state
myristoylation or palmitoylation of eNOS, suggesting that additional
mechanisms are involved in the control of the subcellular location of
eNOS. It might be possible that hypochlorite-modified LDL leads to
changes in the physicochemical properties of membranes similar to that
induced by oxLDL (68) or even gives rise to oxidative damage of certain
cholesterol-rich membrane compartments. These alterations may lead to
instabilities of eNOS interaction with proteins or lipids of Golgi or
plasma membrane and mislocation to other membrane compartments.
Reactive oxygen species, for example, have been shown to reduce the
association of eNOS with caveolin-1 in caveolae-enriched membranes
(69). A decreased interaction of eNOS with caveolin-1 as measured by coimmunoprecipitation experiments was also found after pretreatment of
cells with NaOCl-LDL suggesting a disruption of the caveolin-eNOS regulatory cycle under these conditions and an eNOS movement
independent of caveolin-1.
Recently, Blair et al. (36) and Uittenbogaard et
al. (58) demonstrated that in addition to acylation the
cholesterol content of caveolae is important for subcellular eNOS
location and NO formation and may be a target for the regulation of NO
synthesis by oxLDL. In these studies endothelial cells expressing eNOS
exclusively in their plasma membrane were incubated for 1 h with
Cu2+-oxidized LDL which led to a complete loss of
caveolae-associated cholesterol and to a subsequent translocation of
both eNOS and caveolin-1 from the plasma membrane to internal membranes
(not Golgi or endoplasmic reticulum) (36). High density lipoproteins prevented the effects of oxLDL by preserving caveolar cholesterol content (58). The experimental design of these studies is different from our experimental approach including the cellular model, the type
of LDL modification, and the incubation time. Furthermore, in contrast
to our study oxLDL was not internalized into the cells. Thus, although
our findings agree with the studies by Blair et al. (36) and
Uittenbogaard et al. (58) regarding the translocation of
eNOS to internal membranes, the mechanisms underlying the eNOS redistribution may be different. Indeed, NaOCl-LDL treatment of cells
did not alter the cholesterol content of a plasma membrane-enriched subcellular fraction, suggesting that the mislocalization of eNOS induced by hypochlorite-modified LDL cannot be attributed to
cholesterol depletion. Moreover, our data reveal that cyclodextrin,
which had been shown to deplete the plasma membrane of cholesterol and to disrupt caveolae (59), induced a redistribution of eNOS that was
different from the one seen after preincubation of cells with NaOCl-LDL. Cyclodextrin caused a displacement of both eNOS and caveolin-1 from the plasma membrane, thus confirming that cholesterol is essential for eNOS incorporation into the plasmalemmal compartment (36, 58) but did not modify the perinuclear localization of eNOS that
was clearly affected by NaOCl-LDL. Interestingly, despite the drastic
effect of cyclodextrin on the translocation of plasmalemmal eNOS, this
compound did not inhibit agonist-induced endothelial NO formation. In
contrast, agonist-induced NO synthesis in cyclodextrin-treated cells
was even slightly increased, which may probably be due to a
dissociation of eNOS from inhibiting factors such as caveolin-1. Taken
together, our data point to an important contribution of Golgi-located
eNOS to overall NO production in HUVEC since an inhibition of cellular
NO formation was only observed when both a disintegration of
Golgi-located eNOS and a reduction of plasmalemmal eNOS occurred.
In summary, the present findings demonstrate that
hypochlorite-modified LDL inhibits agonist-induced endothelial NO
formation in a time- and concentration-dependent manner.
The decreased NO formation was neither due to substrate nor cofactor
deficiencies nor to changes in eNOS expression but was paralleled by an
intracellular redistribution of eNOS. Our data confirm the importance
of specific intracellular membrane targeting for eNOS activity and
indicate that Golgi-located eNOS may significantly contribute to
overall NO formation in HUVEC. Based on these in vitro
findings we propose that hypochlorite-modified LDL may promote
endothelial dysfunction in vivo thereby being involved in
early atherogenic processes.