© 1997 American Heart Association, Inc.
Articles |
Inhibition of Inducible Nitric Oxide Synthase Restores Endothelium-Dependent Relaxations in Proinflammatory Mediator-Induced Blood Vessels
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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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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; CaCl2, 1.6; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 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 expression29 ), cycloheximide (20 µg/mL, an inhibitor of protein synthesis), N-
-tosyl-L-lysine chloromethyl ketone (TLCK;
100 µmol/L, an inhibitor of iNOS
expression30 ), and pyrrolidine dithiocarbamate (PDTC;
100 µmol/L, another inhibitor of iNOS
expression29 ) 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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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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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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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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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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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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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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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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Discussion |
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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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature.. 1980;288:373-376.[Medline] [Order article via Infotrieve]
2. Siney L, Lewis MJ. Endothelium-derived relaxing factor inhibits platelet adhesion to cultured porcine endocardial endothelium. Eur J Pharmacol.. 1992;229:223-226.[Medline] [Order article via Infotrieve]
3. Scott-Burden T, Vanhoutte PM. The endothelium as a regulator of vascular smooth muscle proliferation. Circulation.. 1993;87:V51-V55.
4.
de Lanerolle P, Nishikawa M, Yost DA, Adelstein RS.
Increased phosphorylation of myosin light chain
kinase after an increase in cyclic AMP in intact smooth muscle.
Science.. 1984;223:1415-1417.
5.
Kilbourn RG, Belloni P.
Endothelial cell production of nitrogen
oxides in responses to interferon gamma in combination with tumor
necrosis factor, interleukin-1, or endotoxin. J Natl
Cancer Inst.. 1990;82:772-776.
6.
Scott-Burden T, Schini VB, Elizondo E, Junquero DC,
Vanhoutte PM. Platelet-derived growth factor suppresses and
fibroblast growth factor enhances cytokine-induced
production of nitric oxide by cultured smooth muscle
cells. Circ Res.. 1992;71:1088-1100.
7.
Thorin-Trescases N, Hamilton CA, Reid JL, McPherson
KL, Jardine E, Berg G, Bohr D, Dominiczak AF. Inducible
L-arginine/nitric oxide pathway in human internal mammary artery and
saphenous vein. Am J Physiol.. 1995;268:H1122-H1132.
8.
Xie QW, Kashiwabara Y, Nathan C. Role of
transcription factor NF-B/Rel in induction of nitric oxide
synthase. J Biol Chem.. 1994;269:4705-4708.
9. Mülsch A, Bassenge E, Busse R. Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and calcium-independent mechanism. Naunyn Schmiedebergs Arch Pharmacol.. 1989;340:767-770.[Medline] [Order article via Infotrieve]
10. Robert R, Chapelain B, Jean T, Neliat G. Interleukin-1 impairs both vascular contraction and relaxation in rabbit isolated aorta. Biochem Biophys Res Commun.. 1992;182:733-739.[Medline] [Order article via Infotrieve]
11.
Aoki N, Siegfried M, Lefer AM. Anti-EDRF effect
of tumor necrosis factor in isolated, perfused cat carotid
arteries. Am J Physiol.. 1989;256:H1509-H1512.
12.
Julou-Schaeffer G, Gray GA, Fleming I, Schott C,
Parratt JR, Stoclet JC. Loss of vascular responsiveness induced
by endotoxin involves L-arginine pathway. Am J
Physiol.. 1990;259:H1038-H1043.
13. Bosaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine-5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest.. 1987;79:170-182.
14.
Freiman PC, Mitchell GG, Heistad DD, Armstrong ML,
Harrison DG. Atherosclerosis impairs
endothelium-dependent vascular relaxation to
acetylcholine and thrombin in primates. Circ Res.. 1986;58:783-789.
15.
Parker JL, Adams HR. Selective inhibition of
endothelium-dependent vasodilator capacity by
Escherichia coli endotoxemia. Circ Res.. 1993;72:539-551.
16.
Zeiher AM, Drexler H, Wollschlager H, Just H.
Endothelial dysfunction of the coronary
microvasculature is associated with impaired coronary blood
flow regulation in patients with early
atherosclerosis. Circulation.. 1991;84:1984-1992.
17.
Verbeuren TJ, Jordaens FH, Zonnekeyn LL, van Hove CE,
Coene M-C, Herman AG. Effect of
hypercholesterolemia on vascular reactivity in
the rabbit. Circ Res.. 1986;58:552-564.
18.
Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD,
Yeung AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary
vasomotor response to acetylcholine relates to risk factors for
coronary artery disease. Circulation.. 1990;81:491-497.
19. McKenna TM. Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. J Clin Invest.. 1990;86:160-168.
20. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol.. 1990;65:297-302.[Medline] [Order article via Infotrieve]
21. Moyer CF, Sajuthi D, Tulli H, Williams JK. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol.. 1991;138:951-956.[Abstract]
22. Rus HG, Niculescu F, Vlaicu R. Tumor necrosis factor-alpha in human arterial wall with atherosclerosis. Atherosclerosis.. 1991;89:247-254.[Medline] [Order article via Infotrieve]
23. Yoshizumi M, Perrella MA, Burnett JC, Lee M. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res.. 1993;73:205-209.[Abstract]
24. Matsubara T, Ziff M. Increased superoxide anion release from human endothelial cells in response to cytokines. J Immunol.. 1986;137:3295-3298.[Abstract]
25. Busse R, Mülsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett.. 1990;275:87-90.[Medline] [Order article via Infotrieve]
26. Beasley D, Schwartz JH, Brenner BM. Interleukin-1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest.. 1991;87:602-608.
27. Schini VB, Junquero DC, Scott-Burden T, Vanhoutte PM. Interleukin-1 induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta. Biochem Biophys Res Commun.. 1991;176:114-121.[Medline] [Order article via Infotrieve]
28.
Buga GM, Griscavage JM, Rogers NE, Ignarro LJ.
Negative feedback regulation of endothelial cell
function by nitric oxide. Circ Res.. 1993;73:808-812.
29. Schini-Kerth VB, Bara A, Mülsch A, Busse R. Pyrrolidine dithiocarbamate selectively prevents the expression of the inducible nitric oxide synthase in the rat aorta. Eur J Pharmacol.. 1994;265:83-87.[Medline] [Order article via Infotrieve]
30.
Schini-Kerth VB, Boese M, Busse R, Fisslthaler B,
Mülsch A. N-alpha-tosyl-L-lysine
chloromethylketone prevents the expression of the
inducible nitric oxide synthase in vascular smooth muscle by blocking
activation of nuclear factor-B. Arterioscler Thromb
Vasc Biol.. 1997;17:672-679.
31. Southan GJ, Szabo C, Thiemermann C. Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity. Br J Pharmacol.. 1995;114:510-516.[Medline] [Order article via Infotrieve]
32.
Durante W, Schini VB, Scott-Burden T, Junquero DC,
Kroll MH, Vanhoutte PM, Schafer AI. Platelet inhibition by
an L-arginine-derived substance released by IL-1 ß-treated vascular
smooth muscle cells. Am J Physiol.. 1991;261:H2024-H2030.
33.
Mülsch A, Fisslthaler B, Schini V, Busse R.
Activation of the nuclear transcription factor B is a key
mechanism for nitric oxide synthase gene activation in
macrophages and smooth muscle cells. In: Moncada S,
Feelisch M, Busse R, Higgs EA, eds. Biology of Nitric Oxide.
London: Portland Press; 1994:142-145.
34.
Greenberg S, Xie J, Wang Y, Cai B, Kolls J, Nelson S,
Hyman A, Summer WR, Lippton H. Tumor necrosis factor-alpha
inhibits endothelium-dependent relaxation.
J Appl Physiol.. 1993;74:2394-2403.
35.
Parker JL, Keller RS, DeFily DV, Laughlin MH, Novotny
MJ, Adams HR. Coronary vascular smooth muscle function
in E. coli endotoxemia in dogs. Am J Physiol.. 1991;260:H832-H841.
36. Wylam ME, Samsel RW, Umans JG, Mitchell RW, Leff AR, Schumacker PT. Endotoxin in vivo impairs endothelium-dependent relaxation of canine arteries in vitro. Am Rev Respir Dis.. 1990;142:1263-1267.[Medline] [Order article via Infotrieve]
37. Beasley D, Cohen RA, Levinsky NG. Interleukin-1 inhibits contraction of vascular smooth muscle. J Clin Invest.. 1989;83:331-335.
38.
French JF, Lambert LE, Dage RC. Nitric oxide
synthase inhibitors inhibit interleukin-1 ß-induced
depression of vascular smooth muscle. J Pharmacol
Exp Ther.. 1991;259:260-264.
39. Fleming I, Julou-Schaeffer G, Gray GA, Parratt JR, Stoclet JC. Evidence that an L-arginine/nitric oxide dependent elevation of tissue cyclic GMP content is involved in depression of vascular reactivity by endotoxin. Br J Pharmacol.. 1991;103:1047-1052.[Medline] [Order article via Infotrieve]
40.
Martin E, Nathan C, Xie QW. Role of interferon
regulatory factor 1 in induction of nitric oxide synthase.
J Exp Med.. 1994;180:977-984.
41. MacIntyre DE, Bushfield M, Shaw AM. Regulation of platelet cytosolic free calcium by cytosolic nucleotides and protein kinase C. FEBS Lett.. 1985;188:383-388.[Medline] [Order article via Infotrieve]
42. Karaki H, Sato K, Ozaki H, Murakami K. Effects of sodium nitroprusside on cytosolic calcium level in vascular smooth muscle. Eur J Pharmacol.. 1988;156:259-266.[Medline] [Order article via Infotrieve]
43. Takai Y, Kaibuchi K, Matsubara T, Nishizuka Y. Inhibitory action of guanosine 3', 5'-monophosphate on thrombin-induced phosphatidylinositol turnover and protein phosphorylation in human platelets. Biochem Biophys Res Commun.. 1981;101:61-67.[Medline] [Order article via Infotrieve]
44. Rapoport RM. Cyclic guanosine monophosphate inhibition of contraction may be mediated through inhibition of phosphatidylinositol hydrolysis in rat aorta. Circ Res.. 1986;18:407-410.
45. Nakashima S, Tohmatsu T, Hattori H, Okano Y, Nozawa Y. Inhibitory action cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets. Biochem Biophys Res Commun.. 1986;135:1099-1104.[Medline] [Order article via Infotrieve]
46.
Cavallini L, Coassin M, Borean A, Alexandre A.
Prostacyclin and sodium nitroprusside inhibit the activity of
the platelet inositol 1,4,5-triphosphate receptor and promote its
phosphorylation. J Biol Chem.. 1996;271:5545-5551.
47.
Duhe RJ, Nielsen MD, Dittman AH, Villacres EC, Choi EJ,
Storm DR. Oxidation of critical cysteine residues of type I
adenylyl cyclase by o-iodosobenzoate or nitric oxide reversibly
inhibits stimulation by calcium and calmodulin.
J Biol Chem.. 1994;269:7290-7296.
48.
Gopalakrishna R, Chen ZH, Gundimeda U. Nitric
oxide and nitric-generating agents induce a reversible inactivation of
protein kinase C activity and phorbol ester binding.
J Biol Chem.. 1993;268:27180-27185.
49.
Caselli A, Camici G, Manao G, Moneti G, Pazzagli L,
Cappugi G, Ramponi G. Nitric oxide causes inactivation of the
low molecular weight phosphotyrosine protein phosphatase.
J Biol Chem.. 1994;269:24878-24882.
50.
Radomski MW, Palmer RM, Moncada S. An
L-arginine/nitric oxide pathway present in human platelets
regulates aggregation. Proc Natl Acad Sci U S A.. 1990;87:5193-5197.
51. Busse R, Mülsch A. Calcium-dependent nitric oxide synthase in endothelial cytosol is mediated by calmodulin. FEBS Lett.. 1990;265:133-136.[Medline] [Order article via Infotrieve]
52.
Singer HA, Peach MJ. Calcium- and
endothelial-mediated vascular smooth muscle relaxation
in rabbit aorta. Hypertension.. 1982;4:19-25.
53. Lückhoff A, Busse R. Increased free calcium in endothelial cells under stimulation with adenosine nucleotides. J Cell Physiol.. 1986;126:414-420.[Medline] [Order article via Infotrieve]
54.
Shin WS, Sasaki T, Kato M, Hara K, Seko A, Yang
W-D, Shimamoto N, Sugimoto T, Toyo-oka T. Autocrine and
paracrine effects of endothelium-derived relaxing
factor on intracellular Ca2+ of endothelial
cells and vascular smooth muscle cells. J Biol
Chem.. 1992;267:20377-20382.
55.
Bauersachs J, Popp R, Hecker M, Sauer E, Fleming I,
Busse R. Nitric oxide attenuates the release of
endothelium-derived hyperpolarizing factor.
Circulation.. 1996;94:3341-3347.
56.
Graier WF, Myers PR, Rubin LJ, Adams HR, Parker JL.
Escherichia coli endotoxin inhibits agonist-mediated cytosolic
Ca2+ mobilisation and nitric oxide biosynthesis in cultured
endothelial cells. Circ Res.. 1994;75:659-668.
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