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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1746-1755

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1746-1755.)
© 1997 American Heart Association, Inc.

Articles

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.


*    Abstract
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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-{alpha} (1000 U/mL), interferon-{gamma} (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-{alpha}-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


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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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-{alpha} (TNF-{alpha}).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-{kappa}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-{alpha} 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-{alpha} 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.


*    Methods
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 up arrowIntroduction
 *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-{alpha}-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-{alpha} (1000 U/mL), IFN-{gamma} (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-{gamma} were from Sigma; bradykinin was from Bachem Biochemica GmbH (Heidelberg, Germany); recombinant human TNF-{alpha} 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{alpha},9{alpha}-epoxymethano-prostaglandin F2{alpha}) 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).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1aDown and b). In contrast, the endothelium-dependent relaxation evoked by the calcium ionophore A23187 was only slightly reduced by the cytokine treatment (Fig 1cDown). Similarly, exposure of porcine coronary arteries to TNF-{alpha} (1000 U/mL), IFN-{gamma} (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 1Down). 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 2aDown 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-{alpha} (1000 U/mL)-, and IFN-{gamma} (500 U/mL)-Treated Porcine Coronary Artery Rings with Endothelium



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  		Figure 2. Concentration-relaxation curves to sodium nitroprusside (SNP) in (a) control and IL-1 ß-treated endothelium-intact rabbit carotid artery rings constricted to a similar level of tension with phenylephrine and (b) in control and LPS-, TNF-{alpha}, IFN-{gamma}-treated endothelium-intact porcine coronary artery rings constricted to a similar level of tension with U46619 (0.1 to 0.3 µmol/L). Rings were incubated in serum-free culture medium in the presence of either (a) solvent or IL-1 ß (100 U/ml) for 7 hours and (b) polymyxin B (1 µg/mL) or a combination of LPS (10 µg/mL), TNF-{alpha} (1000 U/ml), and IFN-{gamma} (500 U/ml) for 15 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 11 (a) and 7 (b) experiments.

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 3Down).



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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.

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 2Down; Fig 4aDown and b). In the presence of the preferential inhibitor of iNOS,31 S-methylisothiourea (10 µmol/L) maximum contraction to phenylephrine was restored (Fig 4Down, Table 2Down), 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.

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 5aDown). Rings exposed either to IL-1 ß for only 15 minutes (Fig 5aDown) or incubated with IL-1 ß for 7 hours in combination with cycloheximide (20 µg/mL, an inhibitor of protein synthesis; Fig 5bDown) failed to express iNOS (Table 2Up and Reference 3232 ) and relaxed maximally to acetylcholine (Fig 5Down). The cycloheximide treatment alone did not affect the endothelium-dependent relaxation to acetylcholine (Fig 5bDown). 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 6aDown) or PDTC (100 µmol/L; Fig 6bDown) 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 6cDown). All of these treatments have been shown to abolish the induced generation of NO (Table 2Up and References 29 and 3329 33 ). The TLCK, PDTC, and SMT treatment alone only minimally affected the endothelium-dependent relaxation to acetylcholine (Fig 6aDown-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.

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 7aDown 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 7aDown 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.

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 8aDown). In contrast, perfusates from IL-1 ß (100 U/mL for 7 hours)-treated carotid arteries and those from LPS (10 µg/mL)-, TNF-{alpha} (1000 U/mL)-, and IFN-{gamma} (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 8bDown). 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 8bDown). 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-{alpha} (1000 U/mL)-, and IFN-{gamma} (500 U/mL for 15 hours)-treated coronary arteries was associated with a rapid and pronounced relaxation of detector arteries (Figs 8aDown and 9Down 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 8Down and 9Down).



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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).



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  		Figure 9. Cumulative data from bioassay experiments showing the release of NO from acetylcholine (1 µmol/L)-stimulated (a) control and IL-1 ß (100 U/ml)-treated rabbit carotid artery segments and from bradykinin (0.1 µmol/L)-stimulated (b) control and LPS (10 µg/mL)-, TNF-{alpha} (1000 U/mL)-, and IFN-{gamma} (500 U/mL)-treated porcine coronary artery segments. The effect of addition of NG-nitro-L-arginine (0.1 mmol/L) to the perfusate is also shown. The release of NO is expressed as the percentage of the relaxation of the detector ring to glyceryl trinitrate (100 pmol/L). Results are shown as means±SEM of 5 (a) and 4 (b) different experiments. *P<.05 inhibitory effect of NG-nitro-L-arginine.

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 10Down). 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 10Down).



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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 11Down, 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 11Down, 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
up arrowTop
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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-{alpha}, 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-{kappa}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-{kappa}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-{gamma} = interferon-{gamma}
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-{alpha}-tosyl-L-lysine-chloromethyl ketone
TNF-{alpha} = tumor necrosis factor-{alpha}


*    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.


*    References
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 *References
 

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