Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1746-1755
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1746-1755.)
© 1997 American Heart Association, Inc.
Inhibition of Inducible Nitric Oxide Synthase Restores Endothelium-Dependent Relaxations in Proinflammatory Mediator-Induced Blood Vessels
Paul Kessler;
Johann Bauersachs;
Rudi Busse;
;
Valerié B. Schini-Kerth
From Zentrum der Anästhesiologie (P.K.) and Zentrum der Physiologie,
(J.B., R.B., V.B.S.-K.), Klinikum der Johann Wolfgang Goethe-Universität,
Frankfurt am Main, Germany.
Correspondence to V.B. Schini-Kerth, Ph.D., Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail busse{at}merlin.add.uni-frankfurt.de.
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Abstract
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Abstract Endothelium-dependent relaxations
mediated by nitric
oxide (NO) are attenuated in arteries exposed to
proinflammatory
mediators. Because proinflammatory mediators stimulate
the expression
of the inducible NO synthase (iNOS) in vascular cells,
the role
of iNOS-derived NO in the impaired
endothelium-dependent relaxation
was examined in
arterial ring preparations. Exposure of rabbit
carotid
arteries to interleukin-1 ß (IL-1 ß; 100
U/mL for 7 hours) and
porcine coronary arteries to a combination
of tumor necrosis
factor-

(1000 U/mL), interferon-

(500 U/mL),
and
lipopolysaccharide (10 µg/mL) for 15 hours (conditions
that
are associated with iNOS expression) markedly attenuated
relaxations to
receptor-dependent agonists, whereas those to
the calcium ionophore
A23187 and sodium nitroprusside were virtually
unchanged. The impaired
relaxation was not associated with a
reduced level of the constitutive
endothelial NOS (cNOS) but
was accompanied by a reduced
formation of biologically active
NO as assessed in a bioassay system.
The attenuated relaxation
of carotid arteries to acetylcholine was not
affected by superoxide
dismutase and was neither found in arteries
exposed to IL-1
ß for only 15 minutes nor in IL-1 ß-treated
arteries
for 7 hours followed by a 17-hour incubation period without
the
cytokine. Furthermore, no impaired relaxation was found in
rings
exposed to IL-1 ß in combination with either cycloheximide
or
N-

-tosyl-
L-lysine chloromethyl ketone or
pyrrolidine dithiocarbamate,
treatments that prevent iNOS expression.
In addition, selective
inhibition of iNOS with
S-methylisothiourea (10 µmol/L)
completely restored
acetylcholine-induced relaxations.
These findings indicate that the continuous generation of NO
induced by proinflammatory mediators plays a major role in the
inhibition of endothelium-dependent relaxation, most
likely by impairing a step in the signal transduction cascade that
links activation of endothelial receptors to the
calcium-calmodulin-dependent activation of NOS.
Key Words: nitric oxide inducible NOS constitutive NOS isolated blood vessels bioassay system
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Introduction
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Nitric
oxide (NO) regulates a number of physiologic processes
in the vascular
system, including the local control of vascular
tone, platelet
activation, and presumably, the proliferation
of smooth muscle
cells.
1 2 3 In intact vascular segments, NO
is generated
from
L-arginine by the constitutively expressed
NO synthase
(NOS) in endothelial cells. Activation of
endothelial
cells by both receptor-dependent and
-independent agonists results
in the activation of the
calcium-calmodulin-dependent NOS and
a transient generation
of modest amounts of NO.
In addition, NO can be generated in most types of vascular cells after
the expression of the inducible NOS (iNOS) in response to
lipopolysaccharide4 and/or cytokines, such
as interleukin-1 ß (IL-1 ß) and tumor necrosis factor-
(TNF-
).5 6 7 The activity of iNOS is calcium-independent
and appears to be controlled predominantly at the transcriptional level
through the activation of several transcription factors, including
nuclear factor-
B and interferon regulatory factor-1.8 9
Therefore, once expressed, iNOS can generate NO at a maximal rate over
long periods of time.
Besides stimulating the expression of iNOS in vascular cells,
proinflammatory mediators can also affect the generation of NO by the
endothelium. Indeed, the in vitro exposure of rabbit
aorta to IL-1 ß and cat carotid arteries to TNF-
for several hours
is accompanied by an impaired endothelium-dependent
relaxation.10 11 Moreover, a blunted
endothelium-dependent relaxation was found in arteries
isolated from endotoxemic animals or from experimental animal models of
atherosclerosis, in atherosclerotic human arteries, and
also in the coronary circulation of patients with risk factors
for coronary artery disease and proximal atherosclerotic
lesions.12 13 14 15 16 17 18 Because the generation of both IL-1 ß and
TNF-
is upregulated in arteries from endotoxemic animals and at
sites of atherosclerotic lesions,19 20 21 22 these
proinflammatory mediators may affect endothelial
function in endotoxemia and atherosclerosis. However,
the mechanisms by which proinflammatory mediators blunt
endothelium-dependent relaxation have not been
elucidated. Altered endothelial function may reflect a
reduced level of the endothelial NO synthase (cNOS), as
has been suggested by findings obtained in cultured
endothelial cells.23 Alternatively,
because cytokines markedly increase the generation of
oxygen-derived radicals in cultured endothelial
cells,24 the impaired
endothelial-dependent relaxations may reflect an
excessive inactivation of NO by superoxide anions. In addition, the
ability of cytokines to stimulate the expression of iNOS with
the subsequent release of large amounts of NO in most types of vascular
cells25 26 27 may also contribute to the impaired
endothelial function, because large amounts of NO
inhibited partially purified cNOS and the biosynthesis of NO in bovine
aortic endothelial cells.28 Therefore, the
major aim of the study presented herein was to clarify the role
of iNOS-derived NO in the impaired
endothelium-dependent relaxation in isolated arteries
exposed to proinflammatory mediators.
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Methods
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Preparation of Blood Vessels
Rabbit Aorta and Carotid Arteries
New Zealand White rabbits of both genders (1.4 to 3.2 kg of
body
weight) were anesthesized with sodium pentobarbital (60
mg/kg
intravenous). After exsanguination both carotid arteries
and
the thoracic aorta were removed and placed in Krebs-Henseleit
solution
(mmol/L composition: NaCl, 144; KCl, 5.9;
CaCl
2, 1.6; MgSO
4,
1.2;
KH
2PO
4, 1.2; NaHCO
3, 25;
D-glucose, 11.1) to which the
cyclooxygenase
inhibitor diclofenac was
added at a concentration of 1 µmol/L.
The carotid
arteries were cleaned of adventitial adipose and
connective tissue and
cut into rings of 3-mm length for organ
chamber experiments or segments
of 20-mm length for Western
blot analysis and
immunohistochemistry and of 40-mm length for
bioassay experiments.
Rabbit aortic rings (3 mm) were used for
the detection of NO in
the bioassay experiments. The endothelium
was removed
from aortic rings and from some carotid artery rings
by gently rubbing
the intimal surface with a pair of blunted
forceps. Carotid artery
rings were incubated in 0.5 mL and segments
in 3 mL of culture medium
(minimum essential medium containing
2 mmol/L glutamine,
5 mmol/L
N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic
acid
NaOH, 5 mmol/L
N-hydroxyethyl-piperazine-N'-2-ethanesulfonic
acid
NaOH (pH 7.3), 50 U/mL penicillin, 50 µg/mL streptomycin,
0.1%
bovine serum albumin, and 1 µg/mL polymyxin B)
in the
absence and presence of IL-1 ß (100 U/mL, to induce iNOS
expression
29 ), cycloheximide (20 µg/mL, an
inhibitor of protein synthesis),
N-

-tosyl-
L-lysine
chloromethyl ketone (TLCK;
100 µmol/L, an inhibitor of
iNOS
expression
30 ), and pyrrolidine dithiocarbamate (PDTC;
100
µmol/L, another inhibitor of iNOS
expression
29 ) or a
combination of IL-1 ß and an iNOS
inhibitor for 7 hours
in a cell culture incubator. In
addition, some carotid artery
rings were incubated for only 15 minutes
or 4 hours with IL-1
ß (100 U/mL) or for 7 hours followed by a
17-hour incubation
period in medium without the cytokine.
Porcine Coronary Arteries
Porcine hearts (obtained from a local slaughter house) were
placed immediately into ice-cold Krebs-Henseleit solution and
transported to the laboratory. The coronary arteries were
dissected, cleaned of adventitial adipose and connective tissue, and
cut into rings of 3-mm length for organ chamber studies and segments of
40-mm length for bioassay experiments. The expression of iNOS in
coronary artery rings was elicited similarly to that for
carotid arteries, except that a combination of TNF-
(1000 U/mL),
IFN-
(500 U/mL), and LPS (10 µg/mL) and a 15-hour
incubation period were used.
Organ Chamber Experiments
The carotid and coronary artery rings were suspended
between F30 force transducers (Hugo Sachs Elektronik, March, Germany)
and a rigid support for measurement of changes in isometric force in
10-mL organ chambers (Schuler-Organbad, kindly made available by Hugo
Sachs Elektronik) containing warm (37°C) and oxygenated
(95% O2-5% CO2) Krebs-Henseleit solution.
Passive tension was adjusted over a 30-minute equilibration period to
approximately 2 g for carotid artery rings and 5 g for
coronary artery rings. Thereafter, the carotid artery rings
were constricted with phenylephrine (1 to 3
µmol/L) and the coronary artery rings with the
thromboxane mimetic U46619 (0.1 to 0.3
µmol/L). The lack of relaxation to acetylcholine (1
µmol/L) or bradykinin (0.1 µmol/L) was used to
demonstrate the successful removal of the
endothelium.
After washout, the carotid and coronary artery rings with
endothelium were allowed to equilibrate for 20 minutes
and then constricted again, respectively, with
phenylephrine and U46619 to a similar level of contraction
before a cumulative concentration-relaxation curve to the test
compound. In some experiments, control and IL-1 ß-treated rabbit
carotid artery rings were incubated with either
S-methylisothiourea (SMT; 10 µmol/L, a
concentration that has been shown in preliminary experiments to
selectively inhibit the IL-1 ß-induced hyporeactivity of
endothelium-denuded rings to contractile agents without
affecting the endothelium-dependent relaxation of
intact rings) or superoxide dismutase (0.1 µmol/L) for 30
minutes before being contracted with phenylephrine followed
by a concentration-relaxation curve to acetylcholine.
Superfusion Bioassay Experiments
An aortic ring without endothelium (detector)
was suspended between a GM2/GM3 force transducer (Scaime, Annecy,
France) and a rigid support for measurement of changes in isometric
force and superfused at a flow rate of 2 mL/min with warmed (37°C)
and oxygenated (95% O2-5% CO2)
Krebs-Henseleit solution containing 1 µmol/L diclofenac
and 30 nmol/L superoxide dismutase (direct line). Passive
tension was adjusted over a 30-minute equilibration period to
approximately 2 g. The aortic ring was then constricted with
1 µmol/L phenylephrine, and the absence of
the endothelium was confirmed by the lack of relaxation
to a bolus of acetylcholine (10 nmol). Thereafter, the detector ring
was relaxed with a bolus of glyceryl trinitrate (100 pmol) to test the
sensitivity of the aortic ring.
Both a control and a proinflammatory mediator-treated carotid or
coronary artery segment were cannulated at both ends and
suspended horizontally in parallel in a thermostatic (37°C) organ
chamber containing oxygenated (95% O2-5%
CO2) Krebs-Henseleit solution. The segments were perfused
with Krebs-Henseleit solution at a flow rate of 2 mL/min. After an
equilibration period of 30 minutes, the
endothelium-denuded detector ring was moved from the
direct line underneath the perfusates from donor segments
(delay, approximately 1 second). The release of NO from the
endothelium of donor segments was elicited by
acetylcholine (1 µmol/L) added to the luminal
perfusate of carotid artery segments and by bradykinin
(0.1 µmol/L) added to the luminal perfusate of
coronary artery segments.
Western Blot Analysis of Endothelial cNOS
The rabbit carotid artery segments were rapidly frozen and
homogenized in liquid nitrogen. The proteins were extracted
by ethanol precipitation of the phenol phase obtained during the acid
guanidinium thiocyanate extraction and were subjected to
SDS-polyacrylamide gel electrophoresis (8% gel). The separated
proteins were transferred to nitrocellulose membranes. After blocking
nonspecific binding, nitrocellulose blots were incubated first with a
primary mouse monoclonal antibody against human cNOS (Transduction
Laboratories). After washing and blocking steps, a secondary polyclonal
anti-mouse antibody conjugated to horseradish peroxidase (Amersham
International, Braunschweig, Germany) was added. cNOS immunoreactivity
was visualized by exposing an x-ray film to blots incubated with the
ECL reagent (Amersham). Prestained molecular mass markers (Bio-Rad)
were used as standards for SDS-polyacrylamide gel
electrophoresis immunoblot analysis. The
autoradiographs were analyzed by scanning densitometry (Image
Master, Pharmacia, Germany).
Immunohistochemistry
Carotid artery segments were fixed with formaldehyd (4%, pH
7.4), and then embedded in paraffin. Tissue sections were cut 4-µm
thick. After washing in decreasing solutions of ethanol,
endogenous peroxidase was blocked by immersing slides in
3% hydrogen peroxide in methanol for 10 minutes, followed by washing
in phosphate-buffered saline solution (150 mmol/L NaCl;
100 mmol/L phosphate, pH 7.4; three washes, 10 minutes
each) and then with phosphate-buffered saline solution (PBS) containing
0.2 mg/mL proteinase K (Sigma) at 37°C for 10 minutes.
Sections were incubated at 37°C for 30 minutes with a rabbit
polyclonal iNOS antibody (Alexis Corporation) diluted 1:500 in PBS
containing 0.05% bovine serum albumin and 0.1% sodium azide.
Sections were washed in PBS and successively incubated first with
biotinylated goat antiserum to rabbit immunoglobulin G diluted 1:100
in PBS containing 0.05% bovine serum albumin, then with
biotinylated mouse antiserum to goat immunoglobulin G diluted 1:300,
then with freshly prepared streptavidin-peroxidase for 30 minutes, and
finally with 3-amino 9-ethyl carbazol (30 µg/mL) in 0.1
mol/L acetate buffer (pH 5.2) with 0.06% hydrogen peroxide at
22°C for 7.5 minutes.
Data Analysis
Unless otherwise indicated, data are expressed as mean±SEM. n
represents the number of arteries studied. The molar
concentration of a vasorelaxant agonist producing a 50% inhibition
(IC50) and of a vasoconstrictor causing a half-maximal
contraction (EC50) was calculated for each
concentration-response curve. In the bioassay system, relaxations
evoked by perfusates from either acetylcholine or
bradykinin-stimulated segments are expressed as a percentage of that
evoked by a bolus of glyceryl trinitrate. Statistical analysis
was performed by Student's t test for paired or unpaired
observations when appropriate, and when more than two treatments were
compared, by a one-way analysis of variance (ANOVA) followed by
a Bonferroni t test for multiple comparisons. A value of
P<0.05 was considered statistically significant.
Materials
IL-1 ß was obtained from Collaborative Research, Inc.
(Bedford, Mass.); acetylcholine, calcium ionophore A23187,
cycloheximide, TLCK, PDTC, sodium nitroprusside, substance P,
phenylephrine, Escherichia coli LPS, serotype
127:B8, and human recombinant IFN-
were from Sigma; bradykinin was
from Bachem Biochemica GmbH (Heidelberg, Germany); recombinant human
TNF-
was from Boehringer-Ingelheim (Ingelheim, Germany);
pentobarbital sodium (Nembutal) was from Sanofi (München,
Germany); Diclofenac (Voltaren injection solution) was from Ciba-Geigy
(Wehr, Germany); NG-nitro-L-arginine
was from Serva (Heidelberg, Germany); glyceryl trinitrate was from
Pohl-Boskamp (Hohenlochstedt, Germany); recombinant SOD (Peroxinorm)
was from Grünenthal (Aachen, Germany); SMT was kindly provided by
Garry Southan, Frederick Cancer Research Center (Frederick, Md); U46619
(9,11-dideoxy-11
,9
-epoxymethano-prostaglandin
F2
) was kindly provided by Upjohn (Ann Arbor, Mich). The
minimal essential medium was purchased from PAN Systems, Chemische
Produkte GmbH (Aidenbach, Germany), and antibiotics were obtained from
Boehringer Mannheim (Mannheim, Germany).
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Results
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Organ Chamber Experiments
In endothelium-intact carotid arteries, the
relaxations evoked
by both acetylcholine and substance P were markedly
attenuated
following exposure to IL-1 ß (100 U/mL for 7 hours; Fig
1a

and b). In contrast, the
endothelium-dependent relaxation
evoked by the calcium
ionophore A23187 was only slightly reduced
by the cytokine
treatment (Fig 1c

). Similarly, exposure of porcine
coronary
arteries to TNF-

(1000 U/mL), IFN-

(500 U/mL), and
LPS (10
µg/mL) for 15 hours caused a marked impairment
of the
relaxation in response to bradykinin, whereas that evoked
by the
calcium ionophore A23187 was not affected (Table 1

).
The treatment with the
proinflammatory mediators affected the
relaxation to the NO donor
sodium nitroprusside neither in the
carotid artery nor in the
coronary artery (Fig 2a

and
b).

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Figure 1. Concentration-relaxation curves to
acetylcholine (a), substance P (b), and calcium ionophore A23187 (c) in
control and IL-1 ß-treated rabbit carotid artery rings constricted to
a similar level of tension with phenylephrine (a: 3.2±0.2
g and 2.9±0.2 g; b: 3.1±0.1 g and 3.0±0.2 g; c: 3.2±0.1 g and
3.0±0.1 g, respectively). Rings were incubated in serum-free culture
medium in the presence and absence of IL-1 ß (100 U/ml) for 7 hours
before the organ chamber assay. All experiments were performed in the
presence of diclofenac (1 µmol/L). Results are shown as
mean±SEM of 10 (a), 8 (b), and 10 (c) experiments. *P<.05
versus control.
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Table 1. IC50 Values and Maximal Relaxations
(Emax) Evoked by Bradykinin and A23187 of Control and LPS
(10 µg/mL)-, TNF- (1000 U/mL)-, and IFN- (500 U/mL)-Treated
Porcine Coronary Artery Rings with Endothelium
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Because cytokines can markedly increase the generation of
superoxide anions in endothelial cells,24
inactivation of nitric oxide by superoxide anions may contribute to the
impaired endothelium-dependent relaxation in
proinflammatory mediator-treated arteries. However, superoxide
dismutase (0.1 µmol/L) affected the
endothelium-dependent relaxation to acetylcholine
neither in control nor in IL-1 ß (100 U/mL for 7 hours)-treated
carotid arteries (Fig 3
).

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Figure 3. Effect of superoxide dismutase on the
concentration-dependent relaxation curves to acetylcholine in control
and IL-1 ß-treated endothelium-intact rabbit carotid
artery rings constricted to a similar level of tension with
phenylephrine. Rings were incubated in serum-free culture
medium in the absence and presence of IL-1 ß (100 U/ml) for 7 hours
and then examined in the organ chamber assay in the presence and
absence of superoxide dismutase (SOD; .1 µmol/L).
Preconstriction levels of tension were 3.3±0.2 g, 3.1±0.1 g, 2.9±0.2
g, and 3±0.2 g in control, SOD-, IL-1 ß-, and SOD- plus IL-1
ß-treated rings, respectively. All experiments were performed in the
presence of diclofenac (1 µmol/L). Results are shown as
means±SEM of eight experiments. *P<.05
inhibitory effect of IL-1 ß and IL-1 ß plus SOD.
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The IL-1 ß-induced synthesis of NO was assessed by the attenuation of
phenylephrine-induced contraction in carotid artery
rings. Exposure of carotid arteries to IL-1 ß (100 U/mL) for 7 hours
was accompanied by a similar shift to the right of the
concentration-contraction curve in endothelium-intact
and denuded carotid artery rings (Table 2
; Fig 4a
and b). In the presence of the preferential inhibitor
of iNOS,31 S-methylisothiourea (10
µmol/L) maximum contraction to phenylephrine was
restored (Fig 4
, Table 2
), confirming that the impaired contraction was
the result of an induced generation of NO predominantly by the vascular
smooth muscle.
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Table 2. Phenylephrine-Induced EC50
Values and Maximal Contractions (Emax) of Rabbit Carotid
Artery Rings in the Absence and Presence of IL-1 ß (100 U/mL) and the
Test Compound
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Figure 4. Effect of S-methylisothiourea on the
concentration-dependent contraction curves to phenylephrine
in both control and IL-1 ß-treated endothelium-intact
(a) and endothelium-denuded (b) rabbit carotid artery
rings. Rings were incubated in serum-free culture medium in the absence
and presence of IL-1 ß (100 U/ml) for 7 hours and than examined in
the organ chamber assay in the presence and absence of SMT (10
µmol/L). All experiments were performed in the presence of diclofenac
(1 µmol/L). Results are shown as mean±SEM of 7 (a) and 8 (b)
experiments. *P<.05 versus control.
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Next, the role of the IL-1 ß-induced generation of NO in the
attenuated endothelium-dependent relaxation was
examined. In addition to the marked impairment of
endothelium-dependent relaxation to acetycholine in
carotid arteries exposed to IL-1 ß (100 U/mL) for 7 hours, a
significant but smaller reduction was found in arteries exposed to IL-1
ß (100 U/mL) for 4 hours (Fig 5a
).
Rings exposed either to IL-1 ß for only 15 minutes (Fig 5a
) or
incubated with IL-1 ß for 7 hours in combination with cycloheximide
(20 µg/mL, an inhibitor of protein synthesis; Fig 5b
) failed to express iNOS (Table 2
and Reference 3232 ) and relaxed
maximally to acetylcholine (Fig 5
). The cycloheximide treatment alone
did not affect the endothelium-dependent relaxation to
acetylcholine (Fig 5b
). In addition, maximal relaxations to
acetylcholine were obtained in carotid arteries exposed to IL-1 ß
(100 U/mL) in combination with either TLCK (100 µmol/L;
Fig 6a
) or PDTC (100
µmol/L; Fig 6b
) and also in IL-1 ß-treated (100 U/mL
for 7 hours) carotid arteries examined in the organ chamber in the
presence of S-methylisothiourea (10 µmol/L;
Fig 6c
). All of these treatments have been shown to abolish the induced
generation of NO (Table 2
and References 29 and 3329 33 ). The TLCK, PDTC,
and SMT treatment alone only minimally affected the
endothelium-dependent relaxation to acetylcholine (Fig 6a
-c).

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Figure 5. a. Time-dependent effect of IL-1 ß on the
concentration-dependent relaxation curve to acetylcholine in
endothelium-intact rabbit carotid artery rings. b.
Effect of cycloheximide (20 µg/ml) on the concentration-dependent
relaxation curves to acetylcholine in both control and IL-1 ß-treated
endothelium-intact rabbit carotid artery rings. Rings
were incubated in serum-free culture medium (a) in the absence
(control) and presence of IL-1 ß (100 U/mL), which was present
either for the entire 7-hour incubation period or added only during the
last 15 minutes or 4 hours of the 7-hour incubation period, and (b) in
the absence and presence of cycloheximide, IL-1 ß (100 U/mL), or a
combination of cycloheximide plus IL-1 ß for 7 hours before the organ
chamber. Preconstriction levels of tension were (a) 3.0±0.2 g,
2.9±0.1 g, 3.0±0.1 g and 2.8±0.2 g in control, IL-1 ß (15
minutes)-, IL-1 ß (4 hours)-, and IL-1 ß (7 hour)-treated rings and
(b) 3.3±0.2 g, 3.2±0.3 g, 2.9±0.2 g, and 3±0.2 g in control,
cycloheximide-, IL-1 ß-, and cycloheximide plus IL-1 ß-treated
rings, respectively. All experiments were performed in the presence of
diclofenac (1 µmol/L). Results are shown as mean±SEM of 6 (a)
and 7 (b) experiments. *P<.05 inhibitory effect
of IL-1 ß.
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Figure 6. Effect of (a) TLCK (100 µmol/L), (b) PDTC
(100 µmol/L), and (c) S-methylisothiourea (10
µmol/L) on the concentration-dependent relaxation curves to
acetylcholine in both control and IL-1 ß-treated
endothelium-intact rabbit carotid artery rings. Rings
were incubated in serum-free culture medium (a) in the absence
(control) and presence of either IL-1 ß (100 U/mL), TLCK, PDTC, or a
combination of IL-1 ß and a modulator for 7 hours before the organ
chamber experiments. Carotid artery rings were exposed to SMT (10
µmol/L) only during organ chamber experiments. Preconstriction levels
of tension were (a) 3.2±0.2 g, 3.3±0.3 g, 2.8±0.2 g, and 3.1±0.1 g
in control, TLCK-, IL-1 ß-, and TLCK plus IL-1 ß-treated rings, (b)
3.1±0.1 g, 3.2±0.2 g, 3.0±0.3 g, and 3.1±0.2 g in control, PDTC-,
IL-1 ß-, and PDTC plus IL-1 ß-treated rings, and (c) 2.9±0.2 g,
3.0±0.2 g, 2.7±0.2 g, and 2.9±0.2 g in control, SMT-, IL-1 ß-, and
SMT plus IL-1 ß-treated rings, respectively. All experiments were
performed in the presence of diclofenac (1 µmol/L). Results are
shown as mean±SEM of 7 (a), 8 (b), and 11 (c) experiments.
*P<.05 inhibitory effect of IL-1
ß.
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To assess whether the IL-1 ß-mediated effect on vascular reactivity
was reversible, endothelium-intact carotid arteries
were incubated with IL-1 ß (100 U/mL) for 7 hours, followed by a
17-hour incubation period in medium without the cytokine. This
treatment was not associated with an attenuation of contraction to
phenylephrine and also not with a blunted relaxation to
acetylcholine (Fig 7a
and b). Incubation
of carotid arteries for 24 hours in the absence of IL-1 ß did not
affect their responsiveness to both phenylephrine and
acetylcholine (Fig 7a
and b).

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Figure 7. Recovery of the IL-1 ß (100 U/mL)-mediated
inhibition of (a) concentration-dependent contraction curves to
phenylephrine and (b) concentration-dependent relaxation
curves to acetylcholine in endothelium-intact rabbit
carotid artery rings. Rings were incubated in serum-free culture medium
in the absence and presence of IL-1 ß (100 U/mL) for either 7 hours
or for 7 hours followed by a 17-hour incubation in fresh culture medium
without IL-1 ß before the organ chamber assay. Preconstriction levels
of tension were (b) 3.1±.1 g, 2.9±0.2 g, 2.7±0.2 g, and 3.0±0.3 g
in control (7 hours), control (24 hours)-, IL-1 ß (7 hours)-, and
IL-1 ß (7 hours followed by a 17-hour incubation period without the
cytokine)-treated rings, respectively. All experiments were
performed in the presence of diclofenac (1 µmol/L). Results are
shown as mean±SEM of 6 (a) and 8 (b) experiments. *P<.05
inhibitory effect of IL-1 ß for 7 hours.
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Superfusion Bioassay Experiments
Perfusates from endothelium-intact donor
segments of either rabbit carotid arteries or porcine coronary
arteries did not affect the tone of detector rabbit aortic rings
without endothelium contracted with
phenylephrine (1 µmol/L; Fig 8a
). In contrast, perfusates from
IL-1 ß (100 U/mL for 7 hours)-treated carotid arteries and
those from LPS (10 µg/mL)-, TNF-
(1000 U/mL)-, and IFN-
(500 U/mL for 15 hours)-treated coronary arteries caused a
small but consistent relaxation of detector arteries
(relaxation of 17.4±3.4%, n=6, and of 14.9±2.7%, n=5, respectively;
Fig 8b
). The proinflammatory mediator-induced relaxing activity of
perfusates was abolished by the addition of
NG-nitro-L-arginine (100
µmol/L) to the donor segment (Fig 8b
). Addition of
acetylcholine (1 µmol/L) to the perfusates of
control and IL-1 ß (100 U/mL for 7 hours)-treated carotid arteries
and of bradykinin (0.1 µmol/L) to the perfusates
of control and LPS (10 µg/mL)-, TNF-
(1000 U/mL)-, and
IFN-
(500 U/mL for 15 hours)-treated coronary arteries was
associated with a rapid and pronounced relaxation of detector arteries
(Figs 8a
and 9
and data not shown).
However, the relaxing activity derived from proinflammatory
mediator-treated arteries was significantly smaller than that from
control arteries; these effects were abolished in the presence of
NG-nitro-L-arginine (100
µmol/L) over donor segments (Figs 8
and 9
).

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|
Figure 8. Original tracings from superfusion bioassay
experiments showing acetylcholine (ACh; 1 µmol/L)-stimulated
release of biologically active NO from control (a) and IL-1 ß-treated
(b) endothelium-intact rabbit carotid artery segments.
Carotid segments were incubated in serum-free culture medium in the
absence (a) and presence (b) of IL-1 ß (100 U/ml) for 7 hours before
the beginning of the experiment. The release of NO from donor segments
was assessed as the relaxation evoked by the perfusates of a
detector rabbit aortic ring without endothelium
contracted with phenylephrine (1 µmol/L). The effect
of a bolus application of glyceryl trinitrate (GTN; 100 pmol) directly
to the detector artery and the effect of
NG-nitro-L-arginine (L-NNA; .1
mmol/L) added to the perfusate are also shown. Experiments were
performed in the presence of diclofenac (1 µmol/L) and
superoxide dismutase (30 nmol/L).
|
|
Expression of cNOS Protein
Western blot analysis using a monoclonal cNOS antibody
directed against a 20.4-kDa protein fragment corresponding to amino
acids 1030 to 1209 of human endothelial cell cNOS
revealed a protein band at about 140 kDa in extracts from
endothelium-intact carotid artery segments (Fig 10
). A similar amount of cNOS was
detected in endothelium-intact carotid artery segments
exposed to either IL-1 ß (100 U/ml) or IL-1 ß in combination with
cycloheximide (20 µg/ml) for 7 hours (Fig 10
).

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|
Figure 10. Immunoblot analysis of
endothelial cNOS in control, IL-1 ß-, and IL-1 ß
plus cycloheximide-treated endothelium-intact rabbit
carotid artery segments. Segments were incubated in serum-free culture
medium in the absence and presence of either IL-1 ß (100 U/mL) or
IL-1 ß in combination with cycloheximide (20 µg/mL) for 7 hours.
Tissue extracts were subjected to SDS-polyacrylamide gel
electrophoresis followed by immunoblot analysis
using a monoclonal antibody against human endothelial
cNOS. The cNOS level in human umbilical vein
endothelial cell (HUVECs) extracts is also shown.
Similar findings were obtained in an additional experiment.
|
|
Immunohistochemistry
Under control conditions, endothelium-intact
rabbit carotid artery segments showed no detectable
immunostaining with the antibody directed against mouse
iNOS (Fig 11
, top). However,
immunostaining was apparent in
endothelium-intact carotid artery segments following
exposure to IL-1 ß (100 U/ml) for 7 hours and was mostly associated
with smooth muscle cells from the tunica media (Fig 11
, bottom).

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|
Figure 11. Immunostaining with a polyclonal
antibody against mouse iNOS in frozen sections from control (top) and
IL-1 ß (bottom)-treated endothelium-intact rabbit
carotid artery segments. Segments were incubated in serum-free culture
medium in the absence and presence of IL-1 ß (100 U/mL) for 7 hours
before being processed for immunohistochemistry. Staining is seen
predominantly in the smooth muscle cells from the tunica media
(arrowheads; x200).
|
|
 |
Discussion
|
|---|
The study presented herein shows that the
endothelial NO production
elicited by
receptor-dependent agonists is attenuated by proinflammatory
mediators
and is associated with the concomitant expression
of iNOS in the
vascular wall. Moreover, treatments that prevent
iNOS expression and/or
its activity can fully restore impaired
endothelium-dependent
relaxation. Hence, the
continuously elevated generation of iNOS-derived
NO seems to impair the
activation of the constitutive endothelial
NOS.
Exposure of isolated arteries to proinflammatory mediators, including
the cytokines IL-1 ß and TNF-
, is associated with a
blunted endothelium-dependent relaxation to various
receptor-dependent agonists, including acetylcholine, bradykinin, and
substance P.10 11 34 An impaired
endothelial function to receptor-dependent stimuli is
also found in arteries removed from endotoxemic
animals12 15 35 36 and, as shown in the present study,
in those exposed in vitro to a combination of endotoxin and
proinflammatory cytokines. However, the treatment with
proinflammatory mediators did not affect the
endothelium-independent relaxation evoked by donors of
NO,11 34 35 indicating that the impaired
endothelium-dependent relaxation is not caused by an
altered guanylyl cyclase-cyclic guanosine 3',5'-monophosphate effector
pathway in the vascular smooth muscle.
In contrast to the receptor-dependent agonists, the treatment with
proinflammatory mediators failed to inhibit the receptor-independent
relaxation to the calcium ionophore A23187,15 34 ruling
out a decreased level of either endothelial NOS or the
substrate/cofactors necessary for the biosynthesis of NO as likely
mechanisms for the impaired endothelial function.
Indeed, the level of cNOS in endothelium-intact carotid
arteries as assessed by Western blot analysis was not changed
by IL-1 ß treatment. In addition, the unaffected relaxation to the
calcium ionophore A23187 suggests that an increased inactivation of NO
such as by superoxide anions, the generation of which can be enhanced
in endothelial cells by
cytokines,24 cannot account for the impaired
endothelium-dependent relaxation. Further evidence for
the lack of involvement of superoxide anions is indicated by the fact
that superoxide dismutase failed to restore impaired
endothelium-dependent relaxations to acetylcholine.
Experiments with the superfusion bioassay system indicated that the
release of NO elicited by receptor-dependent agonists from
proinflammatory mediator-treated arteries was significantly smaller
than that from untreated arteries. Hence, the treatment with
proinflammatory mediators impairs the biosynthesis of NO in
endothelial cells, probably by inhibiting an event in
the receptor mechanisms leading to the activation of the constitutively
expressed NO synthase. In contrast to the 7-hour incubation period,
exposure of carotid arteries to IL-1 ß for only 15 minutes failed to
impair relaxations to acetylcholine. The lack of effect of the
short-term treatment suggests that the impaired
endothelium-dependent relaxation is unlikely to be
caused by the direct effect of IL-1 ß receptor-dependent signal
transduction pathways on the agonist-stimulated biosynthesis of NO in
endothelial cells. Moreover, because the protein
synthesis inhibitor cycloheximide prevented the impairment
of endothelium-dependent relaxation to acetylcholine,
the inhibitory effect seems to involve the expression of a
peptide/protein in the arterial wall.
In addition to the reduced endothelium-dependent
relaxation, exposure of isolated arteries to cytokines and/or
endotoxin for several hours leads to an attenuation of the contractile
response to various stimuli.19 25 37 The impaired
contraction reflects the expression of iNOS and the subsequent
synthesis of substantial amounts of NO for prolonged periods of
time.25 38 39 The activity of iNOS is regulated
predominantly at the transcriptional level and appears to involve
several transcription factors, including necrosis factor-
B and
interferon regulatory factor-1.8 40 Consistent
with previous studies, the induction of iNOS was also demonstrated in
carotid arteries exposed to IL-1 ß for 7 hours, resulting in the
attenuation of phenylephrine-induced contractions, and
by the small but consistent relaxation of detector blood
vessels evoked by the perfusates from IL-1 ß-treated carotid
artery segments. Both of these effects were abolished by
inhibitors of NO synthase. Moreover, iNOS
immunostaining was detected within the carotid artery
wall after IL-1 ß treatment.
NO has been shown to affect several intracellular signaling mechanisms
coupling membrane receptors to biologic responses through the cyclic
guanylic acid effector pathway, in various cell types including
platelets, and the vascular smooth muscle. Among the best
characterized actions are the inhibition of calcium
mobilization,41 42 phosphatidylinositol
turnover,43 44 45 and the activity of the inositol
1,4,5-triphosphate receptor.46 Moreover NO can
inactivate adenylyl cyclase,47 protein kinase
C,48 and phosphotyrosine phosphatases49
through its ability to react with the thiol groups present in the
catalytic site of these enzymes. Thus, it is conceivable that the
impaired endothelium-dependent vasodilation in response
to receptor-dependent agonists is mediated by iNOS-derived NO. To
investigate this hypothesis, arteries were exposed to IL-1 ß in
combination with either TLCK (a serine protease inhibitor)
or PDTC (an antioxidant), both of which have been shown previously to
prevent the IL-1 ß-induced expression of iNOS in vascular smooth
muscle cells and isolated arteries by blocking the activation of
necrosis factor-
B.29 30 33 These inhibitors
not only restored, as expected, the ability of arteries to constrict in
response to phenylephrine but also to dilate in response to
acetylcholine. In addition, the responsiveness of arteries treated with
proinflammatory mediators to both contractile and receptor-dependent
relaxing agonists was fully restored by the NOS inhibitor
S-methylisothiourea used at a concentration that selectively
abolished the activity of iNOS without affecting cNOS. Moreover, the
vasodilatory response to acetylcholine was unaffected in IL-1
ß-treated arteries examined at a time when iNOS was no longer
expressed as indicated by the absence of hyporeactivity to
phenylephrine in the organ chamber. A role for the
iNOS-derived NO in the inhibitory effect of proinflammatory
mediators is also consistent with the fact that the impaired
endothelium-dependent relaxation is a slowly developing
process that requires protein synthesis. Although the iNOS can be
expressed in endothelial cells,5 50 the
major source of NO in arteries exposed to proinflammatory mediators is
most likely the vascular smooth muscle, because the presence of the
endothelium failed to potentiate the IL-1 ß-induced
hyporeactivity to phenylephrine. The biosynthesis of NO by
the cNOS in endothelial cells in response to
receptor-dependent agonists is strictly dependent on the increase in
the intracellular concentration of calcium51 52 53 and
appears to reflect the association of the
calcium-calmodulin complex with the NOS.51 The
feedback inhibition of cNOS-derived NO production by
proinflammatory mediators may involve alteration in calcium signaling
in endothelial cells, because NO either generated by
endothelial cNOS itself or provided exogenously by NO
donors depressed the activator calcium signal in response
to receptor-dependent agonists.54 55 Moreover exposure of
cultured endothelial cells to endotoxin significantly
decreased the biosynthesis of NO and the intracellular calcium response
to both bradykinin and adenosine diphosphate.56
However, whether these actions of endotoxin involve the iNOS-derived
generation of NO still remains to be demonstrated.
In conclusion, the present findings indicate that the
inhibitory effect of proinflammatory mediators on the
endothelial NO synthesis by receptor-dependent agonists
is coincident with the expression of the iNOS in the
arterial wall and that the induced generation of NO
accounts for the impaired endothelial function probably
because of its ability to affect calcium signaling in
endothelial cells. Such a regulatory mechanism may help
to explain the blunted endothelium-dependent
vasodilatory capacity of arteries subjected to a proinflammatory
response, such as in sepsis and in
atherosclerosis.12 13 14 15 16 17 18 This mechanism may
protect the vascular wall from excessive amounts of NO generated by
simultanous activation of iNOS and cNOS, which may be deleterious for
vascular cells. In addition, evidence is presented that the
endothelium fully recovers once the proinflammatory
response subsides.
 |
Selected Abbreviations and Acronyms
|
|---|
| cNOS |
= |
constitutive nitric oxide synthase |
IFN- |
= |
interferon- |
| IL-1 ß |
= |
interleukin-1 ß |
| iNOS |
= |
inducible nitric oxide synthase |
| LPS |
= |
lipopolysaccharide |
| NO |
= |
nitric oxide |
| NOS |
= |
nitric oxide synthase |
| PDTC |
= |
pyrrolidine dithiocarbamate |
| SMT |
= |
S-methylisothiourea |
| TLCK |
= |
N- -tosyl-L-lysine-chloromethyl ketone |
TNF- |
= |
tumor necrosis factor- |
|
 |
Acknowledgments
|
|---|
This work was supported by grants from the Deutsche
Forschungsgemeinschaft
(Schi 399/1-2,3) and the Commission of the
European Communities
(BMH 1-CT 93-1893).
Received August 30, 1996;
accepted March 10, 1997.
 |
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