Am J Physiol Heart Circ Physiol
281: H1469–H1475, 2001.
Endothelial dysfunction is induced by
proinflammatory oxidant hypochlorous acid
CHUNXIANG ZHANG,1 RAKESH PATEL,2,4 JASON P. EISERICH,5 FEN ZHOU,3
STACEY KELPKE,1 WENXIN MA,1 DALE A. PARKS,3,4
VICTOR DARLEY-USMAR,2,4 AND C. ROGER WHITE1,4
1
Departments of Medicine, Vascular Biology and Hypertension Program, 2Pathology,
3
Anesthesiology, and 4Center for Free Radical Biology, University of Alabama at Birmingham;
Birmingham, Alabama 35294; and 5Division of Nephrology, Department of Internal Medicine,
University of California at Davis, Davis, California 95616
Zhang, Chunxiang, Rakesh Patel, Jason P. Eiserich,
Fen Zhou, Stacey Kelpke, Wenxin Ma, Dale A. Parks,
Victor Darley-Usmar, and C. Roger White. Endothelial
dysfunction is induced by proinflammatory oxidant hypochlorous acid. Am J Physiol Heart Circ Physiol 281:
H1469–H1475, 2001.—The myeloperoxidase (MPO)-derived
oxidant hypochlorous acid (HOCl) plays a role in tissue
injury under inflammatory conditions. The present study
tests the hypothesis that HOCl decreases nitric oxide (NO)
bioavailability in the vasculature of Sprague-Dawley rats.
Aortic ring segments were pretreated with HOCl (1–50 ␮M)
followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated
relaxation. In contrast, endothelium-independent relaxation
induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed
by exposure of ring segments to L-arginine but not D-arginine.
In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of
the NO metabolites nitrate (NO3⫺) and nitrite (NO2⫺). The
reduction in total NO metabolite production in bovine aortic
endothelial cells was also reversed by addition of L-arginine.
These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.
nitric oxide; endothelium; smooth muscle
(MPO) is a heme protein synthesized
in granules of neutrophils, monocytes, and macrophages. In response to cell activation, the enzyme is
released in phagocytic vacuoles or into the extracellular space (29). Neutrophil activation also initiates the
assembly of the enzyme NADPH oxidase that generates the oxidants superoxide anion (O2⫺) and H2O2.
MPO catalyzes the oxidation of chloride by H2O2 resulting in the formation of the chlorinating and oxidizing species hypochlorous acid (HOCl) (8, 25). HOCl
avidly reacts with a variety of cellular substrates, including thiols, nucleotides, and amines, to result in
MYELOPEROXIDASE
Address for reprint requests and other correspondence: C. R.
White, Univ. of Alabama at Birmingham, Zeigler Research Bldg. Rm
1046, Birmingham, AL 35294 (E-mail: [email protected]).
http://www.ajpheart.org
enhanced vascular permeability, tissue degradation,
and DNA fragmentation (9–10, 31). Compared with its
parent molecule H2O2, HOCl is 10–20 times more effective in oxidizing proteins (9). Because local concentrations of HOCl in inflamed tissues are estimated to
be as high as 5 mM, there is significant potential for
oxidative tissue injury (13, 34).
Whereas the critical role of HOCl in the host-defense
response has been appreciated for some time, recent
data suggest that HOCl also contributes to vascular
injury associated with acute and chronic inflammatory
diseases, including sepsis, atherosclerosis, reperfusion
injury, and degenerative neurologic disorders (17, 29).
HOCl has been implicated as a mediator of structural
injury under these conditions. In this regard, it was
shown that HOCl contributes to the degradation of
matrix proteins by inhibiting tissue inhibitor of metalloproteinase-I (TIMP-1) and thus increasing the activity of matrix metalloproteinases (7, 32). HOCl also
reduces the activity of ␣1-antiproteinase, the normal
function of which is to inactivate elastase (37). Combined effects of an elevation of elastase and increased
degradation of TIMP-I by HOCl would be to enhance
the breakdown of extracellular matrix proteins. Data
also suggest a role for MPO-derived HOCl in atherogenesis. MPO has been colocalized with macrophages
in human atherosclerotic lesions (20–21), and a recent
report shows that HOCl modifies the apolipoprotein
moiety of low-density lipoprotein, thus enhancing foam
cell formation (20, 38). Furthermore, 3-chlorotyrosine,
a reaction product of tyrosine and HOCl, has been
identified as a marker of MPO-dependent injury in
human atheromas (17).
Neutrophil adhesion and/or the elaboration of neutrophil-derived products may also induce functional
changes in the vasculature (11–12, 14, 23, 27). Increased tissue MPO activity and HOCl formation have
been implicated as mediators of reduced nitric oxide
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
H1469
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Received 14 March 2001; accepted in final form 7 June 2001
H1470
HOCL INHIBITS NO FUNCTION
(NO) bioactivity, but the mechanism(s) underlying this
inhibitory response is incompletely understood (5, 16,
24). Herein, data are presented showing that HOCl
inhibits the endothelium-dependent relaxation of rat
aortic ring segments. It is hypothesized that HOCl
reduces NO bioavailability by converting endogenous
L-arginine into an inactive substrate for the endothelial NO synthase (NOS III) isoform.
MATERIALS AND METHODS
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Materials. Acetylcholine (ACh), A23187, sodium nitroprusside (SNP), L-methionine, L-arginine, D-arginine, L-NAME,
phenylephrine (PE), and sodium hypochlorite were obtained
from Sigma Pharmaceuticals; nitrate/nitrite and NOS activity assay kits were from Calbiochem, L-[3H]arginine was
obtained from DuPont NEN, and a monoclonal NOS III
antibody was from Transduction Laboratories. HOCl concentration was determined by monitoring the absorbance of
hypochlorite at 292 nm (⑀ ⫽ 350 M⫺1 䡠 cm⫺1) in 0.1 N NaOH
using a Beckman Diode Array Spectrophotometer model DU
7000.
Animals. Ten-week-old male Sprague-Dawley rats were
obtained from Harlan Breeding Laboratories (Indianapolis,
IN). All rats were maintained at constant humidity (60 ⫾
5%), temperature (24 ⫾ 1°C), and light cycle (6 AM to 6 PM)
and were fed a standard rat pellet diet (Ralston Purina Diet)
ad libitum. All protocols were approved by the Institutional
Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for
the Care and Use of Laboratory Animals (NIH publication
85-23, Revised 1985).
Vessel reactivity studies. Isometric tension was measured
in isolated aortic ring segments of Sprague-Dawley rats.
After the rat was killed, the aorta was excised and cleansed
of fat and adhering tissue. The vessel was cut into individual
ring segments (2–3 mm in width) and suspended from a
force-displacement transducer in a tissue bath. Ring segments were bathed in Krebs-Henseleit buffer of the following
composition (mM): 118 NaCl, 4.6 KCl, 27.2 NaHCO3, 1.2
KH2PO4, 1.2 MgSO4, 1.75 CaCl2, 0.03 Na2EDTA, and 11.1
glucose. Buffer was maintained at 37°C and aerated with
95% O2-5% CO2. A passive load of 2 g was applied to all ring
segments and maintained at this level throughout the experiment. At the beginning of each experiment, indomethacintreated ring segments were depolarized with KCl (70 mM) to
determine the maximal contractile capacity of the vessel.
Rings were then thoroughly washed with Krebs-Henseleit
buffer and allowed to equilibrate.
In subsequent experiments, vessels were submaximally
contracted (50% of KCl response) with PE (⬃3 ⫻ 10⫺8 to 10⫺7
M). When tension development reached a plateau, ACh (10⫺9
to 3 ⫻ 10⫺6 M) or the calcium ionophore A23187 (10⫺9 to
10⫺5 M) were added cumulatively to the bath to evoke endothelium-dependent relaxation. Whereas ACh stimulates calcium-dependent NO formation in response to a ligand-receptor interaction, A23187 bypasses cell membrane-bound
receptors and acts as a calcium-permeable pore. In other
experiments, endothelium-independent relaxation was
tested by the cumulative addition of the NO donor SNP.
Vasoconstrictor responses were tested by cumulative addition of PE. In some experiments, ring segments were pretreated with HOCl (1–50 ␮M) for 1 h, followed by thorough
rinsing. The residual effects of HOCl treatment on functional
responses of aortic ring segments were then tested by addition of ACh, A23187, or SNP. In some experiments, the HOCl
scavenger L-methionine (50 ␮M) was concurrently added
with HOCl. In other experiments, rings were exposed to
HOCl and washed, followed by incubation with L-arginine (1
mM) or D-arginine (1 mM) for an additional 30 min. In
related experiments, ring segments were pretreated with
L-arginine or D-arginine in the absence of HOCl. Real time
data were collected for all experiments and downloaded to an
IBM PC for analysis using WorkBench PC for Windows
(DASYTECH version 3, Strawberry Tree). Dose-response
profiles for different experimental conditions were analyzed
and tested for differences in relaxation parameters.
Cell culture. Bovine aortic endothelial cells (BAECs) were
isolated from aortas obtained from a local abattoir. BAECs
were maintained in medium 199 containing 5% fetal bovine
serum, 5% iron-supplemented calf serum, 10 ␮M thymidine,
and penicillin-streptomycin. Low-passage (subcultures 4–7)
BAECs were serum deprived 18 h before the study.
Measurement of endothelial cell NOS III protein and activity. Effects of HOCl pretreatment on NOS III protein were
assessed by Western blot. BAECs were preincubated with
HOCl (1–50 ␮M) for 1 h, followed with rinsing. Cells were
homogenized in lysis buffer containing 1% Triton X-100 and
protease inhibitors in Tris-buffered saline (pH 7.5). The protein was then denatured by boiling. Approximately 100 ␮g of
protein from each sample was separated on a 6% SDSpolyacrylamide gel and transferred to nitrocellulose. The
nitrocellulose membrane was blocked for 60 min with 5% dry
milk and 0.01% Tween-20 in Tris-buffered saline. The blots
were incubated overnight with primary monoclonal NOS III
antibody (1:2,000 dilution). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amersham). Autoradiograms exposed in the linear range of film
density were scanned and analyzed using a Fluorchem Digital Imaging System (Alpha Innotech).
NOS III activity was monitored in membrane fractions of
BAECs by measuring the conversion of L-[3H]arginine to
L-[3H]citrulline. Serum-deprived cells were exposed to HOCl
(1–50 ␮M) for 1 h, followed by extensive washing. Cells were
then scraped from culture flasks, collected, and centrifuged
(10,000 rpm/30 s). An aliquot (10 ␮l) of the pellet containing
membrane-associated NOS III protein was resuspended in
phosphate-buffered saline (PBS) containing 6 ␮M tetrahydrobiopterin, 10 mM NADPH, 2 ␮M flavin adenine dinucleotide (FAD), 2 ␮M flavin mononucleotide (FMN), 1 ␮M CaCl2,
and 25 ␮Ci/ml L-[3H]arginine (DuPont NEN). Samples were
incubated for 1 h, after which time endogenous NOS III
activity was blocked by the addition of 5 mM EDTA. Reaction
samples were then incubated with ion exchange resin that
binds with positively charged L-[3H]arginine. Aliquots of this
reaction mixture were transferred to spin cups and placed in
microcentrifuge tubes. Tubes were centrifuged (10,000 rpm)
for 30 s to separate neutrally charged L-[3H]citrulline from
the resin-bound L-[3H]arginine. The radioactivity associated
with the resin fraction and the eluant was determined by
scintillation counting. Data were normalized to protein content and are expressed as the percent conversion of
L-[3H]arginine to L-[3H]citrulline.
Measurement of endothelial cell NO synthesis. NO production in BAECs was assessed by monitoring the formation of
the NO metabolites nitrate (NO3⫺) and nitrite (NO2⫺). BAECs
were pretreated with HOCl (0–50 ␮M) in serum-free media
for 1 h, followed by washing. In some experiments, the HOCl
scavenger L-methionine (50 ␮M) was concurrently added in
the presence of 50 ␮M HOCl. In other experiments, BAECs
were exposed to HOCl (50 ␮M) for 1 h, followed by rinsing
and replacement with media containing 1 mM L-arginine for
an additional 30 min. As controls, BAECs were pretreated
with either L-arginine or L-methionine in the absence of
HOCL INHIBITS NO FUNCTION
H1471
tions were analyzed and tested to determine differences in
relaxation responses using the SigmaStat statistical analysis
program. Unpaired observations were assessed by ANOVA
and post hoc testing using the Student-Newman-Keuls test.
RESULTS
Fig. 1. Concentration-dependent effects of hypochlorous acid (HOCl)
on endothelium-dependent relaxation. A: rat aortic ring segments
were incubated with 1 (■, n ⫽ 7), 5 (Œ, n ⫽ 5), 10 (, n ⫽ 7), or 50 ␮M
(}, n ⫽ 8) HOCl for 60 min. Saline vehicle was added to control ring
segments (E, n ⫽ 11). Tissues were then thoroughly washed to
remove unreacted HOCl, and residual effects of the treatment on
vessel function were assessed. Ring segments were submaximally
contracted with phenylepinephrine (PE) followed by cumulative addition of the endothelium-dependent vasodilator ACh. HOCl inhibited ACh-induced relaxation in a concentration-dependent manner.
B: concurrent incubation of ring segments with 50 ␮M L-methionine
(䊐, n ⫽ 7) completely blocked the inhibitory effect of 50 ␮M HOCl (})
on vessel relaxation. Response to ACh in ring segments treated with
50 ␮M L-methionine alone (E, n ⫽ 7) was similar to that of saline
vehicle controls. Data are means ⫾ SE. *Significant difference (P ⬍
0.05) compared with saline control.
HOCl. Cells were then exposed to the calcium ionophore
A23187 (1 ␮M) for 2 h. Superoxide dismutase (200 U/ml) was
added to the incubation medium to reduce cellular superoxide. Aliquots of media were sampled at the end of this period.
NO3⫺ present in the conditioned media was enzymatically
reduced to NO2⫺ by treatment with Escherichia coli-enriched
nitrate reductase. Total NO2⫺ was used as an index of NO
production and was detected using the fluorophore 2,3-diaminonaphthalene (Calbiochem). Under alkaline conditions,
NO2⫺ converts 2,3-diaminonaphthalene to the fluorescent
compound 1(H)-naphthotriazole. Nitrite concentration was
monitored by the spectrofluorometric excitation of 1(H)naphthotriazole at 360 nm and emission at 450 nm. A standard curve was constructed for NaNO2 (0.1–1,000 nM).
Statistical analysis. All results are expressed as means ⫾
SE. Dose-response profiles for different experimental condiAJP-Heart Circ Physiol • VOL
Treatment of rat aortic ring segments with HOCl
(1–50 ␮M) resulted in a concentration-dependent inhibition of ACh-mediated relaxation (Fig. 1). This effect
was persistent because the blunted response to ACh
was maintained after the removal of HOCl from the
tissue bath by extensive washing. The maximum relaxation (Rmax) induced by 3 ␮M ACh decreased progressively with increasing concentration of HOCl (Fig.
Fig. 3. HOCl does not inhibit endothelium-independent relaxation.
Rat aortic ring segments were incubated with HOCl at concentrations ranging from 0 (E, n ⫽ 7); 1 (■, n ⫽ 5); 10 (Œ, n ⫽ 6), and 50 ␮M
(, n ⫽ 6) for 60 min. Endothelium-independent vasodilator sodium
nitroprusside (SNP) was then added to PE-contracted ring segments
in a cumulative fashion. Preincubation with HOCl did not affect
SNP-induced relaxation. Data are means ⫾ SE.
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Fig. 2. HOCl inhibits relaxation induced by A23187. Rat aortic ring
segments were incubated with 1 (■, n ⫽ 6), 5 (Œ, n ⫽ 6), 10 (, n ⫽ 8),
and 50 ␮M (}, n ⫽ 5) HOCl for 60 min. Saline vehicle was added to
control ring segments (E, n ⫽ 9). Tissues were then thoroughly
washed, and residual effects of HOCl on vessel function were tested.
Ring segments were submaximally contracted with PE, followed by
cumulative addition of the endothelium-dependent vasodilator
A23187. HOCl inhibited A23187-induced relaxation in a concentration-dependent manner. Data are means ⫾ SE. *Significant difference (P ⬍ 0.05) compared with saline control.
H1472
HOCL INHIBITS NO FUNCTION
A23187, SNP-induced relaxation, which is mediated by
the vascular smooth muscle cell metabolism of SNP
and concomitant release of free NO, was unaffected by
HOCl treatment (Fig. 3). In additional experiments,
the contractile sensitivity of rat aortic ring segments to
PE was tested in HOCl-treated vessels to determine
whether the diminished vasodilator response to ACh
was related to an increased sensitivity of ring segments to vasoconstrictor stimuli. HOCl did not affect
the sensitivity of aortic ring segments to PE (not
shown).
To gain insight into the mechanism(s) by which endothelial dysfunction is induced, we monitored the
effects of HOCl on the NO synthetic pathway. In initial
experiments, NOS III protein was quantified in HOCltreated BAECs. Densitometric analysis of immunoblots showed that prior exposure to HOCl did not result
Fig. 4. HOCl does not influence nitric oxide synthase
(NOS) III protein or activity. Bovine aortic endothelial
cells (BAECs) were treated with 0, 10, and 50 ␮M HOCl
for 1 h, followed by rinsing with serum-free medium
199. Western blot analysis (A, top) shows no loss of NOS
III protein in BAECs exposed to HOCl at concentrations up to 50 ␮M. Densitometric analysis of bands
from 3 gels are depicted below the blot. B: NOS III
enzymatic activity under these HOCl incubation conditions. Membrane fraction containing NOS III protein
was isolated from BAECs that were previously treated
with 1, 10, or 50 ␮M HOCl. In control experiments, an
equivalent volume of saline vehicle or 1 mM NG-nitroL-arginine methyl ester (L-NAME) was added to BAEC
monolayers. Whereas control experiments showed that
L-NAME inhibited NOS III activity by 70%, no effect of
HOCl was noted. Data are means ⫾ SE (n ⫽ 5 for each
treatment group). *Significant difference (P ⬍ 0.05)
compared with the L-NAME control group.
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1). There was a strong inverse correlation (R ⫽ ⫺0.94;
P ⬍ 0.001) between HOCl concentration and Rmax.
Concurrent incubation of ring segments with the HOCl
scavenger L-methionine (50 ␮M) completely blocked
the inhibitory effect of HOCl (50 ␮M) on vessel relaxation (Fig. 1). ACh-induced relaxation in vessels
treated with L-methionine (50 ␮M) alone was similar to
that of saline vehicle-treated controls. In related experiments, HOCl-treated ring segments were exposed to
the receptor-independent vasodilator A23187 (Fig. 2).
Relaxation induced by the calcium ionophore A23187 is
dependent on endothelial NO production (30). HOCl
inhibited the response of ring segments to A23187 in a
concentration-dependent manner.
In other studies, the endothelium-independent vasodilator SNP was added to HOCl-treated ring segments.
In contrast to responses observed with ACh and
HOCL INHIBITS NO FUNCTION
H1473
Fig. 6. L-Arginine reverses the inhibitory effect of HOCl on endothelium-independent relaxation. A: rat aortic ring segments were incubated with 10 ␮M HOCl for 1 h and washed. Tissues were then
incubated with saline vehicle (, n ⫽ 7), 1 mM L-arginine (■, n ⫽ 9),
or 1 mM D-arginine (F, n ⫽ 7) for 30 min. Tissues were then
contracted with PE, and ACh dose-response experiments were performed. L-Arginine, but not D-arginine, completely reversed the inhibitory effect of HOCl on ACh-induced relaxation. The vasodilator
response to ACh in control rats (E, n ⫽ 11) is included for comparison.
B: in the absence of HOCl, ACh-induced relaxation in ring segments
pretreated with 1 mM L-arginine (䊐, n ⫽ 7) or 1 mM D-arginine (E,
n ⫽ 9) was similar to that of saline vehicle controls (ƒ, n ⫽ 9). Data
are means ⫾ SE. *Significant difference (P ⬍ 0.05) compared with
saline vehicle- and L-arginine-treated ring segments.
Fig. 5. HOCl inhibits the formation of endothelial cell NO metabolites. NO production in BAECs was assessed by monitoring the
formation of the NO metabolites nitrate (NO3⫺) and nitrite (NO2⫺).
Data are expressed as total NO2⫺. BAECs were pretreated with HOCl
(0–50 ␮M) in serum-free media for 1 h, followed by washout. NO2⫺
formation was stimulated by exposure of BAECs to the calcium
ionophore A23187 (1 ␮M) for 2 h. Total NO2⫺ formation was monitored in media samples using the fluorophore 2,3-diaminonaphthalene. HOCl inhibited NO2⫺ formation in a concentration-dependent
manner. The effect of 50 ␮M HOCl was prevented by concurrent
inhibition with 50 ␮M L-methionine (L-meth). Addition of L-arginine
(L-arg) after treatment with HOCl also reversed the inhibitory effect
of 50 ␮M HOCl. Control experiments showed no effect of either
⫺
L-arginine or L-methionine on A23187-stimulated NO2 formation in
the absence of HOCl. Data are means ⫾ SE (n ⫽ 12–18 for each
treatment group). *Significant difference (P ⬍ 0.05) compared with
saline vehicle treatment. #Significant difference (P ⬍ 0.05) compared
with 50 ␮M HOCl treatment.
AJP-Heart Circ Physiol • VOL
formation in BAECs pretreated with either L-methionine or L-arginine was similar to that of saline vehicle
controls. In a final series of experiments, the effects of
supplemental L-arginine on endothelium-dependent
relaxation were tested in HOCl-treated aortic ring segments. Addition of L-arginine completely reversed the
inhibitory effect of HOCl on ACh-induced relaxation.
Under the same treatment conditions, D-arginine was
without effect (Fig. 6).
DISCUSSION
MPO-derived HOCl plays an important role in structural tissue injury under conditions of inflammation
and ischemia-reperfusion (7, 9–10, 31–32). Previous
studies also suggest a correlation between vascular
MPO-HOCl content and a reduction of NO bioavailability (24, 35–36). In this regard, it was shown that
infusion of HOCl into the guinea pig coronary circula-
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in a loss of NOS III protein (Fig. 4). Effects of HOCl on
NOS III activity were assessed using an L-arginine to
L-citrulline conversion assay. Because NOS III protein
is localized in the cell membrane, we isolated membrane fractions of control and HOCl-treated BAECs.
Aliquots of this fraction were added to PBS containing
physiological concentrations of calcium, critical NOS
III cofactors (NADPH, FAD, FMN, and tetrahydrobiopterin), and L-[3H]arginine (25 ␮Ci/ml). L-[3H]arginine
and L-[3H]citrulline were then separated on ion exchange media. In control experiments, L-[3H]arginine
was converted to L-[3H]citrulline and inhibited by NGnitro-L-arginine methyl ester. Earlier incubation with
HOCl did not affect L-[3H]arginine conversion in isolated membrane fractions (Fig. 4).
The residual effects of HOCl on total NO2⫺ formation
were monitored in A23187-stimulated BAECs. Over a
2-h treatment period, 1 ␮M A23187 stimulated an 85%
increase in total NO2⫺ above baseline. Pretreatment of
BAECs with HOCl resulted in a concentration-dependent decrease in A23187-stimulated NO2⫺ formation
(Fig. 5). The maximum inhibitory effect of HOCl on
total NO2⫺ formation was blocked by concurrent incubation with L-methionine (50 ␮M). In some experiments, BAECs were treated with HOCl for 1 h and
then rinsed with fresh media containing 1 mM L-arginine. Addition of L-arginine also prevented the inhibitory effect of HOCl on NO2⫺ formation in A23187stimulated cells. In the absence of HOCl, NO2⫺
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HOCL INHIBITS NO FUNCTION
AJP-Heart Circ Physiol • VOL
found that addition of L-arginine to aortic ring segments after prior exposure to HOCl restored endothelium-dependent relaxation. In contrast, addition of
D-arginine under these conditions was without effect.
Whereas L-arginine is clearly a substrate for NO production in endothelial cells, D-arginine does not react
with NOS III. These results suggest that the ability of
endogenous L-arginine to serve as a substrate for NOS
III is compromised by prior exposure to HOCl. The
restoration of ACh-induced relaxation, which occurs
with L-arginine supplementation, is likely due to an
increase in functional levels of L-arginine in the endothelial cell.
Reactions of HOCl with ␣-amino acids are well documented (18, 19). Specifically, activated neutrophils
use MPO-derived HOCl to convert ␣-amino acids into
reactive aldehydes (19). This proceeds through a series
of reactions in which the ␣-amino acid is first converted
to an ␣-amino-monochloramine. A reactive carbonyl
intermediate is formed which then undergoes molecular rearrangement to form the corresponding aldehyde.
This reaction pathway can be blocked by catalase demonstrating a dependence on HOCl formation (19). Reactive aldehydes play an important role in tissue injury
by covalently modifying proteins (1). Previous data
support the biochemical modification of L-arginine as a
component of endothelial dysfunction. In this respect,
it was shown that methylation of L-arginine compromises NO production and contributes to the pathogenesis of inflammatory cardiovascular disease (3, 22, 28).
It is hypothesized that the defective relaxation induced
by HOCl in the current studies is also related to a
modification of endogenous L-arginine. We recently
found that HOCl reacts with L-arginine to form chlorinated metabolites that possess similar pharmacological
properties as traditional NOS inhibitors (unpublished
observation). HOCl may thus convert endothelial L-arginine into a new product that binds to NOS III in a
reversible manner and acts as a competitive inhibitor
of the enzyme. Clearly, additional studies are required
to identify mechanisms underlying the HOCl-dependent inhibition of endothelial cell function.
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-54815, HL-67930, and HL-03812.
REFERENCES
1. Anderson MM, Requena JR, Crowley JR, Thorpe SR, and
Heinecke JW. The myeloperoxidase system of human phagocytes generates N epsilon-(carboxymethyl) lysine on proteins: a
mechanism for producing advanced glycation end products at
sites of inflammation. J Clin Invest 104: 103–113, 1999.
2. Auchere F and Capeillere-Blandin C. NADPH as a co-substrate for studies of the chlorinating activity of myeloperoxidase.
Biochem J 343: 603–613, 1999.
3. Boger RH, Bode-Boger SM, Szuba A, Tsa PS, Chan JR,
Tangphao O, Blaschke TF, and Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Circulation 98: 1842–1847, 1998.
4. Boger RH, Bode-Boger SM, Phivthong-ngam L, Brandes
RP, Schwedhelm E, Mugge A, Bohme M, Tsikas D, and
Frolich JC. Dietary L-arginine and alpha-tocopherol reduce
vascular oxidative stress and preserve endothelial function in
281 • OCTOBER 2001 •
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tion significantly reduces basal blood flow (24). Under
these conditions, coronary vasodilation in response to
ACh, bradykinin, and adenosine was abolished in vivo
(24). Results of the current studies show that HOCl
also impairs in vitro functional responses of rat arterial
ring segments by inhibiting ACh-mediated relaxation.
Additionally, the impairment of NO function was prevented by concurrent incubation of HOCl-treated vessels with the scavenger L-methionine. The inhibitory
response to HOCl was persistent because it was maintained after the oxidant was removed from the tissue
bath. Vasodilation elicited by the calcium ionophore
A23187 was similarly inhibited by HOCl. These data
suggest that HOCl treatment did not modify binding
interactions between ACh and muscarinic receptors,
but rather interfered with the signaling processes involved in the calcium-dependent synthesis of NO. In
contrast, SNP-mediated relaxation was not altered by
HOCl, suggesting that the “machinery” required for
vessel relaxation was fully intact. Collectively, these
data point to the endothelium as a critical site of HOCl
action.
To gain insight into mechanism(s) underlying HOCldependent endothelial dysfunction, we assessed interactions between the oxidant and the NOS III synthetic
pathway. A recent report suggests that the HOCldependent chlorination of NADPH alters the ability of
the cofactor to support NADPH-dependent enzyme activity (2). Because NOS III activity requires NADPH, it
is possible that the HOCl-dependent modification of
this cofactor may limit the activity of NOS III. In the
current studies, treatment of BAECs with HOCl did
not result in loss of NOS III protein or activity. In these
experiments, enzyme activity was measured in isolated
membrane fractions in buffer that was replete with
NOS III cofactors and substrates. HOCl treatment,
however, significantly reduced formation of the NO
metabolites NO2⫺ and NO3⫺ in A23187-stimulated
BAECs. This response was reversed by exposure of
BAECs to L-arginine. Results of these cellular studies
are consistent with other mechanisms, including the
possibility that HOCl limits the availability of L-arginine, the substrate for NOS III.
Addition of L-arginine to ring segments or cultured
BAECs in the absence of HOCl did not enhance AChinduced relaxation or NO2⫺ formation, respectively.
This suggests that supplemental L-arginine does not
enhance NO production in substrate replete blood vessels or endothelial cells. Results of previous studies
suggest that L-arginine depletion/modification contributes to the development of endothelial dysfunction in
models of inflammatory vascular disease (26, 28). The
impaired endothelium-dependent relaxation, characteristic of isolated blood vessels from hypercholesterolemic rabbits, can be reversed by the addition of exogenous L-arginine to tissues in vitro (4, 6). L-Arginine
similarly protects vascular function in acute inflammatory models that are characterized by neutrophil adhesion and infiltration in the vessel wall (15, 33). Results
of the present studies also point to endogenous
L-arginine as a potential target of HOCl action. We
HOCL INHIBITS NO FUNCTION
5.
6.
7.
8.
9.
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
AJP-Heart Circ Physiol • VOL
22. Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, and
Cooke JP. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation 99: 3092–3095, 1999.
23. Jorge PA, Osaki MR, de Almeida E, Dalva M, and Credidio
Neto L. Endothelium-dependent coronary flow in ischemia
reperfusion. Exp Toxicol Pathol 49: 147–151, 1997.
24. Leipert B, Becker BF, and Gerlach E. Different endothelial
mechanisms involved in coronary responses to known vasodilators. Am J Physiol Heart Circ Physiol 262: H1676–H1683, 1992.
25. Marcinkiewicz J. Nitric oxide and antimicrobial activity of
reactive oxygen intermediates. Immunopharmacology 37: 35–
41, 1997.
26. Maxwell AJ and Cooke JP. Cardiovascular effects of L-arginine. Curr Opin Nephrol Hypertens 7: 63–70, 1998.
27. McKenna R, Laskey RE, Wang Y, Jaeschke H, and
Mathews WR. Effect of endotoxin-enhanced hepatic reperfusion
injury on endothelium-dependent relaxation in rat aorta. Shock
6: 106–111, 1996.
28. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S,
Okuda S, and Imaizumi T. Endogenous nitric oxide synthase
inhibitor. A novel marker of atherosclerosis. Circulation 99:
1141–1146, 1999.
29. Nauseef WM. Insights into myeloperoxidase biosynthesis from
its inherited deficiency. J Mol Med 76: 661–668, 1998.
30. Piepot HA, Boer C, Groeneveld AB, Van Lambalgen AA,
and Sipkema P. Lipopolysaccharide impairs endothelial nitric
oxide synthesis in rat renal arteries. Kidney Int 57: 2502–2510,
2000.
31. Schraufstatter IU, Browne K, Harrisa A, Hyslop PA, Jackson JH, Quehenberger O, and Cochrane CG. Mechanisms of
hypochlorite injury of target cells. J Clin Invest 85: 554–562,
1990.
32. Shabani F, McNeil J, and Tippett L. The oxidative inactivation of tissue inhibitor of metalloproteinase-1 (TIMP-1) by hypochlorous acid (HOCI) is suppressed by anti-rheumatic drugs.
Free Radic Res 28: 115–123, 1998.
33. Sheridan BC, McIntyre RC Jr, Meldrum DR, and Fullerton DA. L-Arginine attenuates endothelial dysfunction in endotoxin-induced lung injury. Surgery 125: 33–40, 1999.
34. Stief TW, Kurz J, Doss MO, and Fareed J. Singlet oxygen
inactivates fibrinogen, factor V, factor VIII, factor X, and platelet
aggregation of human blood. Thromb Res 97: 473–480, 2000.
35. Vinten-Johansen J, Zhao ZQ, Nakamura M, Jordan JE,
Ronson RS, Thourani VH, and Guyton RA. Nitric oxide and
the vascular endothelium in myocardial ischemia-reperfusion
injury. Ann NY Acad Sci 874: 354–370, 1999.
36. Wang P, Ba ZF, Cioffi WG, Bland KI, and Chaudry IH.
Salutary effects of ATP-MgCl2 on the depressed endotheliumdependent relaxation during hyperdynamic sepsis. Crit Care
Med 27: 959–964, 1999.
37. Whiteman M, Szabo C, and Halliwell B. Modulation of peroxynitrite- and hypochlorous acid-induced inactivation of ␣1antiproteinase by mercaptoethylguanidine. Br J Pharmacol 126:
1646–1652, 1999.
38. Yang CY, Gu ZW, Yang M, Lin SN, Garcia-Prats AJ, Rogers
LK, Welty SE, and Smith CV. Selective modification of apoB100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro. J Lipid Res 40: 686–698, 1999.
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017
11.
hypercholesterolemic rabbits via different mechanisms. Atherosclerosis 141: 31–43, 1998.
Campbell B, Chuhran C, and Lefer AM. Vascular endothelial
growth factor attenuates trauma-induced injury in rats. Br J
Pharmacol 129: 71–76, 2000.
Cooke JP. Is atherosclerosis an arginine deficiency disease?
J Investig Med 46: 377–380, 1998.
Desrochers PE and Weiss SJ. Proteolytic inactivation of ␣-1proteinase inhibitor by a neutrophil metalloproteinase. J Clin
Invest 81: 1646–1650, 1988.
Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman
BA, Halliwell B, and van der Vliet A. Formation of nitric
oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391: 393–397, 1998.
Fliss H. Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants. Mol Cell Biochem 84: 177–
188, 1988.
Folkes LK, Candeias LP, and Wardman P. Kinetics and
mechanisms of hypochlorous acid reactions. Arch Biochem Biophys 323: 120–126, 1995.
Fullerton DA, Eisenach JH, McIntyre RC, Friese RS,
Sheridan BC, Roe GB, Agrafojo J, Banerjee A, and Harken
AH. Inhaled nitric oxide prevents pulmonary endothelial dysfunction after mesenteric ischemia-reperfusion. Am J Physiol
Lung Cell Mol Physiol 271: L326–L331, 1996.
Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 6: 167–178, 1999.
Guo Y, Krumwiede M, White JG, and Wangensteen OD.
HOCl effects on tight junctions of rabbit tracheal epithelium.
Am J Physiol Lung Cell Mol Physiol 270: L224–L231, 1996.
Harken AH. Nitric oxide prevents neutrophil-mediated pulmonary vasomotor dysfunction in acute lung injury. J Surg Res
63:23–28, 1996.
Hayward R and Lefer AM. L-Arginine attenuates endothelial
dysfunction and prolongs survival in rats subjected to traumatic
shock. Endothelium 6: 71–79, 1998.
Hayward R, Scalia R, Hopper B, Appel JZ, and Lefer AM.
Cellular mechanisms of heparinase III protection in rat traumatic shock. Am J Physiol Heart Circ Physiol 275: H23–H30,
1998.
Hazen SL, Crowley JR, Mueller DM, and Heinecke JW.
Mass spectrometric quantification of 3-chlorotyrosine in human
tissues with attomole sensitivity: a sensitive and specific marker
for myeloperoxidase-catalyzed chlorination at sites of inflammation. Free Radic Biol Med 23: 909–916, 1997.
Hazen SL, d’Avignon A, Anderson MM, Hsu FF, and Heinecke JW. Human neutrophils employ the myeloperoxidasehydrogen peroxide-chloride system to oxidize ␣-amino acids to a
family of reactive aldehydes. J Biol Chem 273: 4997–5005, 1998.
Hazen SL, Hsu FF, d’Avignon A, and Heinecke JW. Human
neutrophils employ myeloperoxidase to convert ␣-amino acids to
a battery of reactive aldehydes: a pathway for aldehyde generation at sites of inflammation. Biochemistry 37: 6864–6873, 1998.
Heinecke JW. Mechanisms of oxidative damage of low density
lipoprotein in human atherosclerosis. Curr Opin Lipidol 8: 268–
274, 1997.
Heinecke JW. Pathways for oxidation of low density lipoprotein
by myeloperoxidase: tyrosyl radical, reactive aldehydes, hypochlorous acid and molecular chlorine. Biofactors 6: 145–155,
1997.
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