Effects of homocysteine on endothelial nitric oxide production
Xiaohui
Zhang*,
Hong
Li*,
Haoli
Jin,
Zachary
Ebin,
Sergey
Brodsky, and
Michael S.
Goligorsky
Departments of Medicine and Physiology, State University of New
York, Stony Brook, New York 11794-8152
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ABSTRACT |
Hyperhomocysteinemia (HHCy) is an independent and graded
cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate.
Controversy exists as to the effects of HHCy on nitric oxide (NO)
production: it has been shown that HHCy both increases and suppresses
it. We addressed this problem by using amperometric electrochemical NO
detection with a porphyrinic microelectrode to study responses of
endothelial cells incubated with homocysteine (Hcy) to the stimulation
with bradykinin, calcium ionophore, or L-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 µM) resulted in a gradual decline in responsiveness of endothelial cells to the
above stimuli. Hcy did not affect the expression of endothelial nitric
oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and
immunoblotting of proteins modified by tyrosine nitration. Hcy did not
directly affect the ability of recombinant eNOS to generate NO, but
oxidation of sulfhydryl groups in eNOS reduced its NO-generating
activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme
nor formation of nitrotyrosine-modified proteins. In addition, a
scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by
endothelial cells. In conclusion, electrochemical detection of NO
release from cultured endothelial cells demonstrated that concentrations of Hcy >20 µM produce a significant indirect
suppression of eNOS activity without any discernible effects on its
expression. Folates, superoxide ions, and peroxynitrite scavengers
restore the NO-generating activity to eNOS, collectively suggesting
that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.
endothelial cells; nitric oxide synthase; peroxynitrite; nitrotyrosine
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INTRODUCTION |
HYPERHOMOCYSTEINEMIA
(HHCy) is an independent and graded cardiovascular risk factor, the
strength of which is equal to that of hyperlipidemia (4, 15,
31). HHCy is highly prevalent in patients with chronic
renal failure (3) and mortality rate in patients with
homocysteine (Hcy) levels above 20 µM is about sevenfold greater than
in those with plasma Hcy below 9 µM. In a recent study of patients
with end-stage renal disease on hemodialysis, HHCy was a universal
finding, which was independently associated with the past history of
cardiovascular events (19). In these patients HHCy has
been demonstrated to confer a graded, independent, increased risk for
thrombosis of vascular access, either graft or fistula
(33). Therefore, understanding the mechanism(s) of HHCy-induced vascular complications represents a high-priority task.
Endothelial cell dysfunction is a common denominator for a
variety of cardiovascular diseases ( 2, 5, 12, 13, 20, 22, 29, 30, 45).
It has recently been appreciated that altered function of endothelial
nitric oxide synthase (eNOS) and/or decreased availability of nitric
oxide (NO) can account for a broad array of clinical manifestations in
patients with endothelial dysfunction (8, 10, 14, 18, 28, 32, 35,
43). This notion points to eNOS as a potential target for HHCy.
Indeed, some recent data strongly suggest that Hcy acts on eNOS.
Controversy exists, however, as to the effects of HHCy on NO
production: it has been shown that HHCy upregulates (38)
and downregulates it (9). We revisited this problem by
using amperometric electrochemical NO detection with a porphyrinic
microelectrode and studied responses of microvascular endothelial
cells, exposed to increasing concentrations of Hcy, to the stimulation
with bradykinin, calcium ionophore, or L-arginine.
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MATERIALS AND METHODS |
Materials.
DL-Hcy was purchased from Fluka Chemie (Buchs,
Switzerland); 5-methyltetrahydrofolic acid (disodium salt),
L-arginine (free base),
NG-monomethyl-L-arginine acetate
(L-NMMA), bradykinin (acetate salt), calcium ionophore
A-23187, and diamide were purchased from Sigma (St. Louis, MO); and
ebselen and manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP)
were purchased from Alexis (San Diego, CA). NO electrode calibration
was performed by using nitric oxide gas (Praxair, Danbury, CT). Cell
culture materials (culture dishes, serological pipettes, and
polypropylene conical tubes) were obtained from Becton Dickinson
Labware (Lincoln Park, NJ); endothelial cell basal medium-2 was from
Clonetics (Walkersville, MD); and basal medium Eagle, for electrode
testing, was obtained from Life Technologies (Grand Island, NY).
Cell culture.
Microvascular endothelial cells (RMVEC) were previously established and
characterized by our laboratory (37). These
SV-40-immortalized cells were established from explant cultures of
microdissected rat renal resistance arteries and have been shown to
express receptors for acetylated low-density lipoprotein,
immunodetectable von Willebrand antigen, and are capable of capillary
tube formation (37). Human umbilical vein endothelial
cells (HUVEC) were obtained from Clonetics and used between
passages 3 and 8. Both cell types were grown in
endothelial cell basal medium-2 (Clonetics) containing 2% fetal bovine
serum, 100 µg/ml penicillin, and 50 µg/ml streptomycin.
Measurement of eNOS activity with NO-selective microelectrodes in
vitro.
The NO concentration was monitored with porphyrin-electroplated,
nafion-coated, carbon-fiber electrodes (30 µm OD), which were
manufactured as previously detailed (36). The electrode oxidation current was low-pass filtered at 0.5 Hz and sampled every
2 s. The measurements were made by using constant potential amperometry (0.7 mV) utilizing a highly sensitive potentiostat (InterMedical, Nagoya, Japan). Calibration of the electrode was performed before each experiment by using dilutions of freshly prepared
NO-saturated Krebs-Ringer solution, as illustrated in Fig.
1.

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Fig. 1.
Calibration of nitric oxide (NO)-selective
microelectrode. A typical recording of electrode currents
(A) elicited by the addition of known concentration of
dissolved NO (amperometric titration) and linear regression analysis of
the data (B; standard curve, r2 = 0.998) are shown. The sensitivity of the NO electrode is defined as
an increment in current per unitary change in NO concentration (in this
particular case, it was 21.3 pA/10 nM NO).
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For the determination of NO production by the cultured cells, HUVEC
were incubated in the presence of different concentrations of Hcy added
to the culture medium for 24 h. Cells were placed on the stage of
an inverted microscope equipped with a micromanipulator and enclosed in
a Faraday chamber. After a stable baseline current was recorded, cells
were stimulated with bradykinin, A-23187, or L-arginine, as
specified in RESULTS. When necessary, L-NMMA, at the final concentration of 0.2 or 2 mM, was added to the solution to
verify the NO dependence of a recorded electrode current.
Alternatively, recordings were performed at 0.4-mV electrode holding
potential, which is unfavorable for NO determination. Using these
techniques, in the preliminary studies we found no evidence that any of
the utilized agents interferred with the NO electrode function. Before and at the completion of experiments, electrode function was tested with different dilutions of NO-saturated deionized water.
Expression of eNOS and nitrotyrosine.
Confluent HUVEC, treated with various concentrations of Hcy with or
without 5-methyltetrahydrofolate (5-MTHF) (42) or ebselen (1) for 24 h, were lysed in a buffer containing (in
mM) 150 NaCl, 1 sodium orthovanadate, 50 Tris · HCl (pH 8.0), 1 EDTA, 1 EGTA, and 1 dithiothreitol as well as 1% NP-40 and proteinase inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Lysates were cleared by centrifugation (3,000 g for 10 min at
4°C). After protein determination using a bicinchoninic acid kit
(Pierce, Rockford, IL), samples were denatured by boiling in 2.5%
2-mercaptoethanol for 3 min. Twenty micrograms of each sample were run
on 4-20% Tris-glycine (Novex, San Diego, CA) gel. The proteins
were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and
blocked with 1% casein/PBS for 1 h. Immunoblotting was performed
at room temperature for 2 h with monoclonal anti-eNOS antibody at
1:1,000 dilution (Transduction Laboratories, Lexington, KY), followed by peroxidase-conjugated sheep anti-mouse IgG at 1:2,000 dilution. Membranes were developed by using SuperSignal chemiluminescence substrate (Pierce), and the intensity of staining was quantified by
densitometry. Immunodetection of nitrotyrosine expression was performed
according to manufacturer's instructions (Upstate Biotechnology). Briefly, membranes were extensively washed, blocked in freshly prepared
1% casein/PBS at room temperature for 20 min, and incubated with 2 µg/ml of rabbit polyclonal anti-nitrotyrosine overnight at 4°C.
After extensive washing, membranes were incubated with donkey
anti-rabbit IgG (Amershan Life Science) at 1:3,000 dilution in freshly
prepared 1% casein/PBS at room temperature for 1.5 h. Detection
was performed as detailed above. For immunoprecipitation experiments,
500 µg of total lysate were precleared with 15 µl of protein A/G
Plus-agarose beads (Santa Cruz Biotechnology) and the supernatant was
incubated (overnight at 4°C) with 5 µg of monoclonal
anti-nitrotyrosine antibody, followed by precipitation (2 h, 4°C)
with 30 µl of protein A/G Plus-agarose beads, washed in lysis buffer
and boiled with 2× sample loading buffer for 5 min, as described
previously (23). The samples were separated by 4-20%
Tris-glycine gel and immunoblotted with anti-nitrotyrosine antibody as
detailed above.
Measurement of reduced oxygen species.
Reduced oxygen species (ROS) generation by endothelial cells was
investigated by using a nonfluorescent probe 2',7'-dichlorofluorescin (DCFH), which acquires fluorescence properties on ROS-induced oxidation
to DCFH. HUVEC were pretreated with increasing concentrations of Hcy in
the presence or absence of the cell-permeable superoxide dismutase
mimetic MnTBAP (7). After 24 h incubation, HUVEC were
washed with PBS and DCFH was added in its membrane-permeant diacetate
form at a final concentration of 10 µM in the phenol red-free DMEM.
ROS generation by the cells led to oxidation of DCFH, yielding the
fluorescent product dichlorofluorescein (DCF), which was detected at an
emission wavelength of 530 nm (excitation wavelength of 485 nm) by
using a fluorescence plate reader, as described previously
(44).
Recombinant eNOS studies.
eNOS protein, a kind gift from Dr. S. S. Gross, was purified from
Escherichia coli that had been transformed with independent vectors for expression of eNOS and GroELS, as previously described (24). Recombinant eNOS was prepared in 100-µl aliquots
at a concentration of 167 µg/ml (Bradford assay). The activity of a 20-µl sample in 100 µl total volume was 0.416 optical density in
the Griess/nitrite assay, confirming the authenticity of the product. For each experiment 25 pmol eNOS were used. To assess the effects of Hcy, eNOS activity was monitored with an NO-selective microelectrode in a stirred microcuvette at room temperature. The
NO-selective and reference electrodes were equilibrated in an
intracellular buffer at room temperature with constant stirring until a
stable baseline current was obtained. The composition of the buffer was
50 mM Tris · HCl, pH 7.4, 500 µM NADPH, 5 µM FAD, 5 µM
flavin adenine mononucleotide, 100 nM calmodulin, 0.01 mM
CaCl2, and 20 µM L-arginine. One micromolar
tetrahydrobiopterin (BH4) was added to the eNOS aliquots 12-24 h
before the measurements. After a stable baseline was obtained, 25 pmol
eNOS were pipetted into the cuvette and the response was continuously recorded.
Statistical analyses.
Statistical analyses comparing multiple variables were performed by
using ANOVA followed by the Bonferroni correction. For comparisons
between two variables, the unpaired Student's t-test was
used, with a P < 0.05 considered statistically
significant. All values are presented as means ± SE.
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RESULTS |
NO release from endothelial cells stimulated by bradykinin, A-23187
or L-arginine.
Twenty-four-hour preincubation with HCy (10, 20, and 50 µM) did not
cause any detectable phenotypic cell changes but resulted in a gradual
decline in the resposiveness of endothelial cells to the employed
standard stimuli for eNOS (Figs.
2-4).
Responses to bradykinin (10 µM) were affected when
endothelial cells were treated with 10, 20, and 50 µM Hcy producing
58 ± 7, 45 ± 8, and 27 ± 3 nM NO, respectively,
compared with control 53 ± 4 nM NO (this represents
1.7, 21.8, and 52.9% suppression of NO release, respectively) (Fig. 2).
L-Arginine addition (1 mM) to L-arginine-free medium resulted in a rapid release of 276 ± 38 nM of NO;
incubation with 10 µM Hcy decreased this response by 9.8% (249 ± 23.5 nM), 20 µM Hcy produced a 34.1% suppression (181 ± 33 nM), and 50 µM reduced NO production by 83.8% to 44.4 ± 12.1 nM (Fig. 3). Similar results were obtained after 48-h incubation with
Hcy (not shown). A-23187-stimulated release of NO from RMVEC or HUVEC
was affected by 24-h preincubation with Hcy, resulting in a 37.7%
(188.5 ± 57 vs. 303 ± 30 nM in control) and 44.5%
(105.5 ± 18 vs. 190 ± 25 nM in control) suppression at 50 µM Hcy, respectively (Fig. 4, A and B).

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Fig. 2.
Inhibition of bradykinin-stimulated NO release from
endothelial cells by different concentrations of homocysteine (Hcy).
Microvascular endothelial cells (RMVEC) preincubated in the presence of
different concentrations of Hcy for 24 h, with and without 50 µM
5-methyltetrahydrofolate (5-MTHF; MTHF), were stimulated with 10 µM
bradykinin, and NO release was detected with NO-selective
microelectrode. [NO], NO concentration. * Statistically
significant difference from control (CON), P < 0.001 (n = 5-9 experiments for each concentration of
Hcy).
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Fig. 3.
Inhibition of L-arginine-stimulated NO
release from endothelial cells by different concentrations of Hcy.
RMVEC preincubated in the presence of different concentrations of Hcy
for 24 h, with and without 50 µM 5-MTHF, were stimulated with 1 mM L-arginine, and NO release was detected with
NO-selective microelectrode. * Statistically significant difference
from CON, P < 0.001 (n = 6-8
experiments for each concentration of Hcy).
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Fig. 4.
Inhibition of A-23187-stimulated NO release from
endothelial cells by different concentrations of Hcy.
A: human umbilical vein endothelial cells (HUVEC)
preincubated in the presence of different concentrations of Hcy for
24 h were stimulated with 5 µg/ml A-23187, and NO release was
detected with NO-selective microelectrode. * Statistically
significant difference from CON, P < 0.05 (n = 4-5 experiments for each concentration of
Hcy). B: RMVEC preincubated in the presence of 50 µM
Hcy for 24 h, with and without 50 µM 5-MTHF, were stimulated
with 5 µg/ml A-23187, and NO release was detected with NO-selective
microelectrode. * Statistically significant difference from CON,
P < 0.05 (n = 5-13 experiments
for each condition).
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Effects of Hcy on the expression of eNOS.
Western analysis was performed in RMVEC and HUVEC lysates. (The latter
cell type was used due to the poor immunodetection of rat eNOS with the
available antibodies.) Twenty-four-hour incubation in the presence of
10, 20, or 50 µM Hcy did not affect the expression of eNOS (Fig.
5). At neither concentration did Hcy
induce iNOS expression (data not shown).

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Fig. 5.
Expression of endothelial nitric oxide synthase (eNOS) in
HUVEC incubated with different concentrations of Hcy. Western blot
analysis was performed as detailed in METHODS. There was no
suppression of eNOS within this time frame, and addition of 5-MTHF did
not change eNOS abundance. Equal loading was confirmed by stripping of
membranes and reblotting with antibodies against tubulin
(bottom). These experiments were performed twice, with the
same results.
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Lack of a direct effect of Hcy on recombinant eNOS.
To ascertain the possibility of Hcy acting directly on eNOS activity,
in vitro studies on NO generation employing recombinant eNOS were
performed, as previously described (33). The activity of
eNOS was monitored with NO-selective microelectrode before and after
addition of 50 µM Hcy (Fig.
6A). No differences in NO generation existed between two groups (159 ± 21 before vs.
148 ± 43 nM after addition of Hcy; n = 5, P = no significant difference).

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Fig. 6.
Lack of the direct effect of Hcy on NO-generating capacity of a
recombinant eNOS (A) and suppression of NO production by
diamide (B). A: in vitro detection of NO
generation by the recombinant eNOS in the presence and absence of 50 µM Hcy (n = 4-5 experiments for each condition).
eNOS activity was monitored with an NO-selective microelectrode in a
stirred microcuvette at room temperature, as detailed in
METHODS. B: in vitro detection of NO generation
by the recombinant eNOS is suppressed by the diamide-induced oxidation
of sulfhydryl groups in a dose-dependent manner.
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The same experimental setup was used to investigate any potential
effect of ROS, normally generated in the process of Hcy oxidation (see
below and Fig. 7), on eNOS activity.
Oxidation of sulfhydryl groups of eNOS by diamide resulted in a
dose-dependent decrease in NO generation by the recombinant enzyme in
vitro (Fig. 6B). These data indicate that, although Hcy per
se does not directly affect eNOS activity, ROS formed in the process of
Hcy oxidation can decrease the NO-generating activity of eNOS.

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Fig. 7.
Reduced oxygen species (ROS) production in the
Hcy-pretreated HUVEC. Treatment with Hcy (10, 20, and 50 µM for
24 h) resulted in a gradual increase in ROS production, which
reached statistical significance at 50 µM Hcy and was completely
abolished by coincubation with the cell-permeant superoxide dismutase
mimetic manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP; MnT;
40 M). * P < 0.05 vs. CON, n = 6.
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Folate counteracts Hcy inhibition of eNOS.
Because 5-MTHF, a product of methylenetetrahydrofolate reductase,
is required for the remethylation of Hcy to methionine, we next
coincubated endothelial cells growing in high-Hcy medium with
50-100 µM 5-MTHF. After 24 h of coincubation, endothelial cells were stimulated by bradykinin or A-23187 and NO generation was
detected with a NO-selective microelectrode. As shown in Figs. 2-4, coincubation of the cells with 100 µM 5-MTHF restored eNOS responses to the above agonists. This effect was not attributable to
the change in eNOS expression, as combined Hcy and 5-MTHF treatment of
endothelial cells did not result in any changes in the abundance of the
immunodetectable eNOS (Fig. 5).
ROS and nitrotyrosine formation in response to Hcy.
To examine the mechanism of Hcy-induced suppression of NO release from
stimulated RMVEC and HUVEC, we next tested its effect on the generation
of ROS. ROS production was compared in cells pretreated with increasing
concentrations of Hcy using a nonfluorescent probe, DCFH, oxidatively
converted into the fluorescent probe DCF. Incubation of endothelial
cells with 50 µM, but not with 10 or 20 µM, Hcy resulted in a
statistically significant increase in DCF fluorescence (Fig. 7), thus
confirming previous observations on Hcy-induced oxidative stress
(21, 38, 39).
ROS could be also detected by the formation of nitrotyrosine, a marker
of superoxide anion reaction with NO, resulting in peroxynitrite
formation (23). The level of nitrosylation of protein
tyrosine residues was initially evaluated by using Western blot
analysis. All tested concentrations of Hcy resulted in the formation of
nitrotyrosine-modified protein with an apparent molecular mass of 66 kDa. Coincubation with various concentrations of 5-MTHF did not
prevent nitrotyrosine formation (not shown). In a series of experiments
employing immunoprecipitation with monoclonal antibodies against
nitrotyrosine, the resolution of immunodetected proteins modified by
tyrosine nitrosylation has improved over Western analysis (Fig.
8). This improved detection system
allowed us to discern the previously undetectable dose-dependent
increase in nitrotyrosine formation in the presence of escalating
levels of Hcy. Again, 5-MTHF per se increased nitrotyrosine formation
and did not modify Hcy-induced nitrotyrosine formation, thus implying
that 5-MTHF may stimulate ROS formation. Alternatively, these findings
can be attributable to the fact that, by restoring NO generation, 5-MTHF results in the increased supply of NO to participate in the
reaction with superoxide anions to generate peroxynitrite. In contrast,
HUVEC incubation in the presence of a scavenger of peroxynitrite,
ebselen, resulted in the reduced nitrotyrosine formation.

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Fig. 8.
Immunodetection of nitrotyrosine in endothelial cells
cultured in the presence of different concentrations of Hcy. HUVEC were
coincubated with Hcy with or without 5-MTHF (100 µM) or ebselen (50 µM) as indicated. After immunoprecipitation (IP) of proteins modified
by tyrosine nitrosylation, selected material was immunoblotted (IB)
with antibodies to nitrotyrosine (NT), as detailed in
METHODS. Several unidentified bands of molecular mass
~60-70 kDa are readily visible. Coadministration of 5-MTHF did
not affect Hcy-induced formation of nitrotyrosine. However,
coincubation with ebselen inhibited Hcy-induced nitrotyrosine
formation. These experiments were performed twice with the same
results. L, position of IgG light chains on the blot; MW, molecular
weight marker.
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Scavengers of peroxynitrite or superoxide restore NO production in
Hcy-treated cells.
The above findings are indicative of the formation of superoxide and
peroxynitrite by Hcy-treated endothelial cells. Therefore, in the next
series of experiments attempts were made to suppress their effect by
scavenging either superoxide anion or peroxynitrite. Endothelial cells
were incubated with escalating concentrations of Hcy in the presence of
either a superoxide dismutase mimetic, MnTBAP, or a peroxynitrite
scavenger, ebselen, and compared with the effect of 5-MTHF. NO release
from the endothelial cells stimulated with 5 µg/ml A-23187 was
studied 24 h later by using NO-selective microelectrodes. As
summarized in Fig. 9, A-C,
neither of these supplements per se affected NO responses to the
calcium ionophore in the intact endothelial cells, but each was capable
of restoring NO responsiveness to Hcy-treated cells.

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Fig. 9.
NO production by Hcy-treated endothelial cells is
restored by the agents accelerating dismutation of superoxide anions
(MnTBAP; A), scavenging peroxynitrite (ebselen;
B), or accelerating metabolic degradation of Hcy and
reduction of dihydrobiopterin (5-MTHF; FA; C). Effects of
MnTBAP (40 µM), ebselen (50 µM) and 5-MTHF (100 µM),
A-C, respectively, on NO production by HUVEC incubated with
Hcy (50 µM) for 24 h and stimulated with 5 µg/ml A-23187 are
shown. * P < 0.05 compared with CON,
n = 5-8 in all experimental groups, and
n = 14 in control.
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DISCUSSION |
The direct measurement of NO release from cultured microvascular
endothelial cells, employing an NO-selective amperometric electrode
detection system, demonstrated that 50 µM Hcy produced a significant
suppression of NO release from endothelial cells stimulated with
various receptor-dependent, transporter-dependent, and
receptor-independent eNOS stimuli. This effect was not mimicked in the
in vitro system where NO generation by a recombinant eNOS was found to
be unaffected by Hcy, indicating that the effect of Hcy is indirect.
However, oxidation of sulfhydryl groups in eNOS reduced NO-generating
activity of the enzyme. Increased levels of nitrotyrosine, an indicator
of the concomitant NO and superoxide radical formation, explains
decreased NO detection as a result of decreased bioavailability, rather
then suppressed production. This conclusion is further supported by the
finding that scavengers of superoxide anions and peroxynitrite, MnTBAP
and ebselen, reduced oxidant stress in endothelial cells, decreased the
formation of nitrotyrosine-modified proteins, and ultimately improved
endothelial cell NO responsiveness to a nonselective stimulus, the
calcium ionophore A-23187. This is the first demonstration of an
indirect inhibitory effect of Hcy on receptor-mediated,
non-receptor-mediated, and L-arginine-stimulated NO release
by endothelial cells. Considering the long-debated role of Hcy in the
development of atherosclerosis (26, 27), our data suggest
that the reduced availability of NO for its physiological targets,
owing to the formation of peroxynitrite and inhibition of eNOS
activity, may lead to endothelial cell dysfunction, thus explaining
development of cardiovascular complications.
The mechanism of development of endothelial cell dysfunction in
hyper-Hcy deserves analysis. Endothelial cells challenged with all
three eNOS stimuli displayed reduction in NO release. This finding
argues that elevated levels of Hcy do not selectively inhibit
receptor-mediated responses (bradykinin); rather, Hcy inhibits
stimulation of eNOS by increased cytosolic calcium level independently
of receptor function (A-23187) and suppresses the L-arginine "paradox." This indicates a global
dysfunction of eNOS. However, Hcy does not suppress the enzyme
directly. Studies with the recombinant eNOS and in vitro detection of
NO production in the presence of Hcy clearly reject this possibility.
Collectively, these findings would suggest that the reduction in NO
release at high levels of Hcy, which do not suppress the expression of eNOS, is a result of either decreased bioavailability of NO through formation of peroxynitrite or decreased activity of the enzyme per se.
The fact that peroxynitrite formation, judged by nitrotyrosine fingerprinting, was found with all tested concentrations of Hcy, even
those that did not suppress endothelial cell ability to generate NO,
casts doubt on the predominant role of the former mechanism.
An understanding of the mechanism(s) of high Hcy-induced suppression of
NO release from endothelial cells can be derived from the results of
studies on combined effects of Hcy and scavengers of superoxide and
peroxynitrite or the active metabolite of folic acid, 5-MTHF. This
latter compound has clearly improved the amplitude of NO responses to
all stimuli, while failing to change the abundance of eNOS or, even
more importantly, the degree of peroxynitrite formation. This finding
strongly suggests that the improved NO release associated with 5-MTHF
is not due to the reduced peroxynitrite formation and improved
bioavailability of NO, thus implying that folate therapy acts by
improving eNOS activity. The following observations support this
conclusion, as schematically summarized in Fig.
10.

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Fig. 10.
Schematic view of the hypothetical mode of Hcy action in
suppressing the NO-generating capacity of eNOS and pharmacological
modulators of this action (see text for details). Arrows, stimulation
of metabolic pathways; , blockade of metabolic
pathways.
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Hcy is a well-known mediator of oxidative stress in endothelial cells
(21). Upchurch et al. (39) have demonstrated
that Hcy does not affect eNOS expression or its gene transcription; rather, it decreases the activity and steady-state mRNA levels of
glutathione peroxidase, leading to the enhanced generation of reduced
oxygen intermediates. Clinical experience demonstrates that
supplementation with folic acid reduces only marginally the level of
Hcy in hemodialysis patients or patients with HHCy (16, 19,
40), despite dramatic increases in the level of folate. In
patients with familial hypercholesterolemia and normal levels of Hcy,
folate supplementation also improved endothelial dysfunction (41,
42). Collectively, these data suggest that the mode of folate's
action transcends its ability to accelerate the metabolism of Hcy. It
has been shown that folates stimulate regeneration of endogenous BH4
from dihydropterin (BH2) (25). In this vein, recent
studies have revealed that eNOS binding of BH2, instead of BH4,
converts eNOS from a NO-generating to a superoxide-generating enzyme
(17). Collectively, these data indicate that the mechanism whereby Hcy reduces NO release is through oxidative stress in endothelial cells, which could result in the conversion of BH4 to BH2
and suppression of the NO-generating activity of eNOS. The potential
ability of folates to promote the regeneration of BH4 can explain, at
least in part, its the observed restoration of the NO-generating
capacity of the enzyme.
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ACKNOWLEDGEMENTS |
The authors are grateful to Dr. S. S. Gross (Cornell Medical
College, Dept. of Pharmacology) for providing recombinant eNOS used in
these studies and Dr. L. Moore (SUNY Stony Brook, Dept. of Physiology
and Biophysics) for help with manufacturing NO electrodes.
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FOOTNOTES |
*
X. Zhang and H. Li contributed equally to this study.
These studies were supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-45462 and DK-52783 (M. S. Goligorsky). X. Zhang was a PhD student in the Department of
Physiology. Z. Ebin was a high school student participating in the
Intel Project.
Address for reprint requests and other correspondence: M. S. Goligorsky, Dept. of Medicine, State Univ. of New York, Stony Brook,
NY 11794-8152 (E-mail: mgoligorsky{at}mail.som.sunysb.edu).
The costs of publication of this
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Received 2 February 2000; accepted in final form 15 June 2000.
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REFERENCES |
1.
Ali, M,
Schlidt S,
Chandel N,
Hynes K,
Schumaker P,
and
Gewertz BL.
Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction.
Am J Physiol Lung Cell Mol Physiol
277:
L1057-L1065,
1999[Abstract/Free Full Text].
2.
Bell, D,
Johns TE,
and
Lopez LM.
Endothelial dysfunction: implications for therapy of cardiovascular diseases.
Ann Pharmacother
32:
459-470,
1998[Abstract/Free Full Text].
3.
Bostom, AG,
and
Lathrop L.
Hyperhomocysteinemia in end-stage renal disease: prevalecne, etiology and potential association to atherosclerotic outcomes.
Kidney Int
52:
10-20,
1997[ISI][Medline].
4.
Boushey, C,
Beresford S,
Omenn G,
and
Motulsky A.
A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intake.
JAMA
274:
1049-1057,
1995[Abstract].
5.
Cines, DB,
Pollak E,
Buck CA,
Loscalzo J,
Zimmerman GA,
McEver RP,
Pober JS,
Wick TM,
Konkle BA,
Schwartz BS,
Barnathan ES,
McCrae KR,
Hug BA,
Schmidt AM,
and
Stern DM.
Endothelial cell in physiology and in the pathophysiology of vascular disorders.
Blood
91:
3527-3561,
1998[Free Full Text].
6.
Clarke, R,
Daly L,
Robinson K,
Naughten E,
Calahane S,
Fowler B,
and
Graham I.
Hyperhomocysteinemia: an independent risk factor for vascular disease.
N Engl J Med
324:
1149-1155,
1991[Abstract].
7.
Day, B,
Shawen S,
Liochev S,
and
Crapo JD.
A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro.
J Pharm Exp Ther
275:
1227-1232,
1995[Abstract].
8.
De Caterina, R,
Libby P,
Peng H,
Thannickal V,
Rajavashisth T,
Gimbrone M,
Shin W,
and
Liao J.
Nitric oxide decreases cytokine-induced endothelial activation.
J Clin Invest
96:
60-68,
1995[ISI][Medline].
9.
De Groote, MA,
Testerman T,
Xu Y,
Stauffer G,
and
Fang FC.
Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium.
Science
272:
414-417,
1996[Abstract].
10.
Dubey, R,
Jackson E,
and
Luscher TF.
Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cells.
J Clin Invest
96:
141-149,
1995[ISI][Medline].
11.
Dudman, N,
Temple S,
Guo X,
Fu W,
and
Perry MA.
Homocysteine enhances neutrophil-endothelial interactions in both cultured human cells and rats in vivo.
Circ Res
84:
409-416,
1999[Abstract/Free Full Text].
12.
Dzau, VJ,
Gibbons GH,
Mann M,
and
Braun-Dullaeus R.
Future horizons in cardiovascular molecular therapies.
Am J Cardiol
80:
33-39,
1997.
13.
Furchgott, RF,
and
Zawadzki JW.
The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.
Nature
288:
373-376,
1980[ISI][Medline].
14.
Garg, UC,
and
Hassid A.
Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.
J Clin Invest
83:
1774-1777,
1989[ISI][Medline].
15.
Graham, IM,
Daly L,
Refsum H,
Robinson K,
Brattstrom L,
and
Ueland PM.
Plasma homocysteine as a risk factor for vascular disease: the European Concerted Action Project.
JAMA
277:
1775-1781,
1997[Abstract].
16.
Hong, SY,
Yang D,
and
Chang S.
Plasma homocysteine, vitamin B6, vitamin B12 and folic acid in end-stage renal disease during low-dose supplementation with folic acid.
Am J Nephrol
18:
367-372,
1998[ISI][Medline].
17.
Jones, C,
Vasquez-Vivary J,
Griscavage J,
Martasek P,
Masters BSS,
Kalyanaraman B,
and
Gross SS.
Fully-reduced pterins prevent superoxide production by eNOS: explanation for the BH4 requirement of NOSs (Abstract).
Acta Physiol Scand
167, Suppl645:
59,
1999.
18.
Kubes, P,
Suzuki M,
and
Granger DN.
Nitric oxide: an endogenous modulator of leukocyte adhesion.
Proc Natl Acad Sci USA
88:
4651-4655,
1991[Abstract].
19.
Kunz, K,
Petitjean P,
Lisri M,
Chantrel F,
Koehl C,
Wiesel ML,
Cazenave JP,
Moulin B,
and
Hannedouche TP.
Cardiovascular morbidity and endothelial dysfunction in chronic haemodialysis patients: is homocyst(e)ine the missing link?
Nephrol Dial Transplant
14:
1934-1942,
1999[Abstract/Free Full Text].
20.
Leibovich, SJ,
Polverini PJ,
Fong TW,
Harlow LA,
and
Koch AE.
Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism.
Proc Natl Acad Sci USA
91:
4190-4194,
1994[Abstract].
21.
Loscalzo, J.
The oxidant stress of hyperhomocyst(e)inemia (Abstract).
J Clin Invest
98:
507,
1996.
22.
Ludmer, PL,
Selwyn AP,
Shook LT,
Wayne RR,
Mudge GH,
Alexander RW,
and
Ganz P.
Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries.
N Engl Med J
315:
1046-1051,
1986[Abstract].
23.
MacMillan-Crow, L,
Crow J,
Kerby J,
Beckman J,
and
Thompson JA.
Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.
Proc Natl Acad Sci USA
93:
11853-11858,
1996[Abstract/Free Full Text].
24.
Martasek, P,
Liu Q,
Liu J,
Roman LJ,
Gross SS,
Sessa WC,
and
Masters BSS
Characterization of bovine endothelial nitric oxide synthase expressed in E. coli.
Biochem Biophys Res Commun
219:
359-365,
1996[ISI][Medline].
25.
Matthews, RG,
and
Kaufman S.
Characterization of the dihydropterin reductase activity of pig liver methylenetetrahydrofolate reductase.
J Biol Chem
255:
6014-6017,
1980[Abstract/Free Full Text].
26.
McCully, KS.
Homocysteine metabolism in scurvy, growth and arteriosclerosis.
Nature
231:
391-392,
1971[ISI][Medline].
27.
McCully, KS,
and
Wilson RB.
Homocysteine theory of arteriosclerosis.
Atherosclerosis
22:
215-227,
1975[ISI][Medline].
28.
Niu, X,
Smith CW,
and
Kubes P.
Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils.
Circ Res
74:
1133-1140,
1994[Abstract].
29.
Quyiumi, AA,
Dakak N,
Andrews N,
Husain S,
Arora S,
Gilligan D,
and
Panza JA.
Nitric oxide activity in the human coronary circulation. Impact of risk factors for coronary atherosclerosis.
J Clin Invest
95:
1747-1753,
1995[ISI][Medline].
30.
Reddy, KG,
Nair R,
Sheehan H,
and
Hodgson J.
Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasound atherosclerosis in patients with risk factors for atherosclerosis.
J Am Coll Cardiol
23:
833-843,
1994[ISI][Medline].
31.
Refsum, H,
Ueland P,
Nygard O,
and
Vollset S.
Homocysteine and cardiovascular disease.
Annu Rev Med
49:
31-62,
1998[ISI][Medline].
32.
Reyes, A,
Porras B,
Chasalow F,
and
Klahr S.
L-Arginine decreases the infiltration of the kidney by macrophages in obstructive nephropathy and puromycin-induced nephrosis.
Kidney Int
45:
1346-1354,
1994[ISI][Medline].
33.
Shemin, D,
Lapane K,
Bausserman L,
Kanaan E,
Kahn S,
Dworkin L,
and
Bostom AG.
Plasma total homocysteine and hemodialysis access thrombosis: a prospective study.
J Am Soc Nephrol
10:
1095-1099,
1999[Abstract/Free Full Text].
34.
Sundquist, T,
Forslund T,
Bengtsson T,
and
Axelsson K.
S-nitroso-N-acetylpenicillamine reduces leukocyte adhesion to type I collagen.
Inflammation
18:
625-631,
1994[ISI][Medline].
35.
Thom, S,
Ohnishi T,
and
Ischiropoulos H.
Nitric oxide released by platelets inhibits neutrophil
2 integrin function following acute carbon monoxide poisoning.
Toxicol Appl Pharmacol
128:
105-110,
1994[ISI][Medline].
36.
Thorup, C,
Jones CL,
Gross SS,
Moore LC,
and
Goligorsky MS.
Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS.
Am J Physiol Renal Physiol
277:
F882-F889,
1999[Abstract/Free Full Text].
37.
Tsukahara, H,
Gordienko D,
Gelato M,
and
Goligorsky MS.
Direct demonstration of the IGF-1-induced nitric oxide production by endothelial cells.
Kidney Int
45:
598-604,
1994[ISI][Medline].
38.
Upchurch, GR,
Welch G,
Fabian A,
Freedman J,
Johnson J,
Keaney J,
and
Loscalzo J.
Stimulation of endothelial nitric oxide production by homocysteine.
Atherosclerosis
132:
177-185,
1997[ISI][Medline].
39.
Upchurch, GR,
Welch G,
Fabian A,
Freedman J,
Johnson J,
Keaney J,
and
Loscalzo J.
Homocysteine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.
J Biol Chem
272:
17012-17017,
1997[Abstract/Free Full Text].
40.
Van Guldener, C,
Janssen M,
and
Lambert J.
No change in impaired endothelial function after long-term folic acid therapy of hyperhomocysteinemia in haemodialysis patients.
Nephrol Dial Transplnt
13:
106-112,
1998[Abstract].
41.
Verhaar, MC,
and
Rabelink TJ.
Future of folates in cardiovascular disease.
Eur J Clin Invest
29:
657-658,
1999[ISI][Medline].
42.
Verhaar, MC,
Wever R,
Kastelein J,
vanDam T,
Koomans H,
and
Rabelink TJ.
5-Methyltetrahydrofolate, an active form of folic acid, restores endothelial function in familial hypercholesterolemia.
Circulation
97:
237-241,
1998[Abstract/Free Full Text].
43.
Von der Leyen, H,
Gibbons G,
Morishita R,
Lewis N,
Zhang L,
Nakajima M,
Kaneda Y,
Cooke J,
and
Dzau VJ.
Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene.
Proc Natl Acad Sci USA
92:
1137-1141,
1995[Abstract].
44.
Yao, Z,
Tong J,
Tan X,
Li C,
Shao Z,
Kim W,
Vanden Hoek T,
Becker L,
Head A,
and
Schumacker PT.
Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes.
Am J Physiol Heart Circ Physiol
277:
H2504-H2509,
1999[Abstract/Free Full Text].
45.
Zeiher, AM,
Drexler H,
Wollschlager H,
and
Just H.
Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis.
Circulation
83:
391-401,
1991[Abstract].
Am J Physiol Renal Fluid Electrolyte Physiol 279(4):F671-F678
0363-6127/00 $5.00
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