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(Hypertension. 1997;29:65.)
© 1997 American Heart Association, Inc.

Research Articles (Issue 1, Part 1)

Endothelin-1 Inhibits Nitric Oxide Synthesis in Vascular Smooth Muscle Cells

Uichi Ikeda; Keiji Yamamoto; Yoshikazu Maeda; Masahisa Shimpo; Toshiko Kanbe; Kazuyuki Shimada

the Department of Cardiology, Jichi Medical School, Minamikawachi, Tochigi, Japan.

Correspondence to Uichi Ikeda, MD, PhD, Department of Cardiology, Jichi Medical School, Minamikawachi-Machi, Tochigi 329-04, Japan. E-mail uikeda@jichi.ac.jp

	Abstract

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We investigated the effects of endothelin-1 on nitric oxide synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of nitric oxide, and the expression of inducible nitric oxide synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1ß (10 ng/mL) for 24 hours caused a significant increase in nitrite production. Endothelin-1 significantly decreased the interleukin-1ß–induced nitrite production by vascular smooth muscle cells in a dose-dependent manner (10^-11 to 10^-8 mol/L). Incubation with interleukin-1ß for 24 hours induced expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells, whereas endothelin-1 showed a suppressive effect on their expressions. Addition of the endothelin type A receptor antagonist BQ-485, but not the endothelin type B receptor antagonist BQ-788, dose-dependently inhibited the effect of endothelin-1. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 hours, the effect of endothelin-1 was abolished. These results indicate that endothelin-1 acts on endothelin type A receptors and inhibits nitric oxide synthesis in interleukin-1ß–stimulated vascular smooth muscle cells at least partially through a protein kinase C–dependent pathway.

Key Words: interleukin-1 • nitrites • nitric-oxide synthase • muscle, smooth

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO) is synthesized from L-arginine by two enzymes. The generation of NO by constitutive, Ca²⁺-dependent NOS (cNOS) from the vascular endothelium plays an important role in the homeostasis of the vascular system. Recently, two isoforms of cNOS have been cloned from rat brain (nNOS, or type I NOS) and vascular endothelium (eNOS, or type III NOS).^{1 2} The inducible, Ca²⁺-independent enzyme (iNOS, or type II NOS) is expressed in target cells such as macrophages, VSMCs, and cardiac myocytes after stimulation with endotoxin and cytokines.^{3 4} NO inhibits platelet aggregation,⁵ VSMC proliferation,⁶ and leukocyte adhesion to endothelial cells.⁷ Therefore, NOS induction in VSMCs may play a role in limiting local vascular injury associated with atherosclerosis.

Endothelins are a family of 21–amino acid peptides that includes ET-1, ET-2, and ET-3 in mammals. ET-1 is the most potent mammalian vasoconstrictor identified to date.⁸ There is recent evidence that ET-1 plasma levels are increased in patients with symptomatic atherosclerosis⁹ and after percutaneous transluminal coronary angioplasty^{10 11} and that ET-1 expression is increased in human atherosclerotic lesions.^{12 13} ET-1, the function of which is coupled to phospholipase C–mediated phosphoinositide hydrolysis and PKC activation, modulates vascular contractility and proliferation and contributes to the pathogenesis of hypertension and atherosclerosis. However, there have been no reports concerning the effects of ET-1 on the production of NO, another modulator of vascular contraction and proliferation, by vascular smooth muscle. Therefore, in the present study, we investigated the effects of ET-1 on NO synthesis in cultured rat VSMCs.

	Methods

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Materials
Human ET-1 was purchased from Peptide Institute Inc. Human recombinant IL-1ß (specific activity, approximately 2x10⁷ U/mg) was a gift from Otsuka Pharmacy, Tokushima, Japan. The ET_A receptor antagonist BQ-485 and ET_B receptor antagonist BQ-788 were purchased from Calbiochem-Novabiochem Japan Ltd. Mouse macrophage iNOS cDNA was a gift from Dr Y. Kawahara (Kobe [Japan] University School of Medicine). Anti-rat iNOS was a gift from Dr H. Esumi (National Cancer Institute, Kashiwa, Japan). PMA was purchased from Sigma Chemical Co. All other chemicals used were of the highest grade commercially available.

Cell Culture
Primary cultures of VSMCs were obtained from the medias of thoracic aortas of Sprague-Dawley rats (200 to 250 g), as described previously.¹⁴ The cells were grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were harvested twice a week by treatment with 0.125% trypsin and were passaged at a 1:3 ratio in 100-mm culture dishes. A typical growth experiment was performed with cultured cells at passages 5 to 10. Cells (3x10⁴/mL) were plated in 24-well or 100-mm culture dishes in DMEM supplemented as described above and allowed to grow to subconfluence, after which they were preincubated in DMEM containing 0.5% FBS supplemented with insulin (5 µg/mL) and transferrin (5 µg/mL) and were used for the experiments described below.

The investigation was performed in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (Her Majesty's Stationery Office, London, UK).

Nitrite Measurement
NO production by cultured cells was determined by measurement of nitrite contents in the culture medium. VSMCs plated in 24-well dishes were incubated in DMEM containing 0.5% FBS at 37°C. Nitrite contents of the culture medium were determined by mixing 500 µL of medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid).¹⁵ Absorbance at 550 nm was measured, and nitrite concentrations were determined by interpolation of a calibration curve of standard sodium nitrite concentrations against absorbance. After washing, cells were dissolved in 0.2 mL of 1% sodium dodecyl sulfate (SDS) and used for protein assay (BCA protein assay kit); bovine serum was used as a standard. Nitrite levels were corrected by protein measurement; data are shown as nanomoles per milligram of protein.

Northern Blot Analysis
Total RNA was extracted from VSMCs plated in 100-mm culture dishes by the acid guanidinium isothiocyanate/phenol/chloroform method, and 20-µg aliquots were subjected to electrophoresis on 1% agarose gels and then transferred onto nylon filters. The blots were hybridized for 24 hours with a random-primed ³²P-labeled mouse macrophage iNOS cDNA probe¹⁶ ; washed twice with an aqueous solution of 150 mmol/L NaCl, 15 mmol/L sodium citrate, and 0.1% SDS at 65°C; and then exposed to Kodak XAR-5 film for 1 to 2 days at -70°C with one intensifying screen.

Assay for iNOS Protein
iNOS protein expression was analyzed by immunoblotting with an antibody to iNOS as described previously.¹⁶ Briefly, VSMCs were lysed in a buffer containing 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1 mol/L dithiothreitol and were sonicated. The homogenates were centrifuged at 100 000g for 20 minutes, and the supernatants (60 µg protein) were subjected to 10% SDS–polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with anti-iNOS for 2 hours followed by peroxidase-labeled donkey anti-rabbit IgG for 1 hour. Peroxidase-labeled proteins were visualized by incubation with peroxidase color development reagents containing the enzyme substrate 3,3'-diaminobenzidine with NiCl₂ as an enhancer.

Statistical Analysis
Data are expressed as mean±SE. For comparisons between multiple groups, we determined the significance of differences between group means by ANOVA using the least significant difference for multiple comparisons. Differences at values of P<.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of ET-1 on IL-1ß–Induced Nitrite Production
IL-1ß (10 ng/mL) stimulated nitrite production by VSMCs in a time-dependent manner (Fig 1). Nitrite accumulation stimulated by IL-1ß was significantly inhibited by simultaneous treatment of cells with ET-1 (10^-9 mol/L). After a 24-hour incubation, the level of IL-1ß–induced nitrite accumulation in the presence of ET-1 was about 60% of that in the absence of ET-1.

View larger version (15K): [in this window] [in a new window]   Figure 1. Time course of nitrite accumulation stimulated by IL-1ß in the presence or absence of ET-1. VSMCs were unstimulated () or stimulated with 10 ng/mL IL-1ß (•) or 10 ng/mL IL-1ß plus 10-9 mol/L ET-1 () for various periods as indicated. Nitrite accumulation in culture medium was measured as described in "Methods"; values were normalized to protein content per dish. Means±SE are shown (n=4).

Fig 2 shows the dose-response effect of ET-1 on nitrite production. ET-1 inhibited IL-1ß–induced nitrite production by VSMCs in a dose-dependent manner (10^-11 to 10^-8 mol/L), whereas ET-1 by itself did not affect the basal level of nitrite production.

View larger version (16K): [in this window] [in a new window]   Figure 2. Dose dependency of the inhibitory effect of ET-1 on nitrite production. VSMCs were incubated for 24 hours with (solid bars) or without (open bars) 10 ng/mL IL-1ß in the presence of various concentrations of ET-1 (10-11 to 10-8 mol/L) as indicated. Nitrite accumulation in culture medium was measured; values were normalized to protein content per dish. Means±SE are shown (n=4). *P<.05, **P<.01, compared with control samples not exposed to ET-1.

Effects of ET-1 on iNOS mRNA and Protein Expressions
We next examined whether ET-1 inhibited IL-1ß–induced nitrite production at mRNA and protein levels. As shown in Figs 3 and 4, unstimulated cells did not express iNOS mRNA or protein. Incubation with IL-1ß (10 ng/mL) for 24 hours induced expression of iNOS mRNA and protein. However, their expression levels were suppressed in the presence of ET-1 (10^-9 mol/L).

View larger version (79K): [in this window] [in a new window]   Figure 3. Expression of iNOS mRNA in VSMCs. Cells were incubated with 10 ng/mL IL-1ß for 24 hours with or without 10-9 mol/L ET-1. Total RNA was size-fractionated by electrophoresis and transferred onto nylon filters. Filters were hybridized with 32P-labeled cDNA probes for iNOS (top) and 18S rRNA (bottom). Lane 1, control; lane 2, IL-1ß; and lane 3, IL-1ß plus ET-1. Two independent experiments yielded indistinguishable results.

View larger version (38K): [in this window] [in a new window]   Figure 4. Effects of ET-1 on iNOS protein accumulation. VSMCs were incubated for 24 hours with 10 ng/mL IL-1ß and 10-9 mol/L ET-1. Cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot analysis. Positions of molecular mass markers are indicated on the right. The iNOS protein band with a molecular mass of about 125-kD is the band above the 116.5-kD marker. Lane 1, control; lane 2, IL-1ß; and lane 3, IL-1ß plus ET-1. Two independent experiments yielded indistinguishable results.

Effects of Endothelin Receptor Antagonists
At least two endothelin receptors, ET_A and ET_B, are expressed in cultured rat VSMCs and mediate the effects of ET-1.¹⁷ We used the ET_A receptor antagonist BQ-485 and the ET_B receptor antagonist BQ-788 (10^-9 to 10^-7 mol/L) to examine the receptor specificity of the effects of ET-1 on nitrite production by VSMCs. As shown in Fig 5, the inhibitory effect of ET-1 on nitrite production was dose-dependently reduced in the presence of BQ-485 but not BQ-788.

View larger version (14K): [in this window] [in a new window]   Figure 5. Effects of endothelin receptor antagonists on nitrite production by VSMCs treated with ET-1. The ETA receptor antagonist BQ-485 () or ETB receptor antagonist BQ-788 (•) (10-9 to 10-7 mol/L) was added to cultures with IL-1ß (10 ng/mL) plus ET-1 (10-9 mol/L) for 24 hours. Data represent percent inhibition of nitrite production by ET-1 in the presence of antagonists. Data are means±SE of four samples.

Involvement of PKC
Previously, it was reported that a PKC-activating phorbol ester, PMA, inhibits nitrite accumulation in IL-1ß–stimulated VSMCs.¹⁸ It is possible that this signaling pathway is involved in the inhibitory effect of ET-1 on nitrite production. We therefore examined the effects of ET-1 in control and PKC-depleted VSMCs. It has been shown that PKC in VSMCs is downregulated by pretreatment with PMA.¹⁹ Thus, cells were exposed to PMA (10^-6 mol/L) in 10% FBS containing DMEM for 24 hours and then incubated in 0.5% FBS containing DMEM with ET-1 (10^-9 mol/L), IL-1ß (10 ng/mL), and/or PMA (10^-7 mol/L) for a further 24 hours. As shown in Fig 6, in control cells not preincubated with PMA, nitrite levels were significantly increased 24 hours after addition of IL-1ß. Addition of ET-1 or fresh PMA reduced nitrite accumulation in IL-1ß–stimulated cells. On the other hand, in VSMCs preincubated with PMA for 24 hours, IL-1ß still increased nitrite levels, but addition of fresh PMA caused no change in nitrite levels, which is consistent with functional depletion of PKC activity. The IL-1ß–induced nitrite levels were not significantly affected by ET-1 in the PKC-depleted cells.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effects of ET-1 on nitrite accumulation in PKC-depleted cells. VSMCs pretreated (open bars) or not treated (solid bars) with PMA (10-6 mol/L) for 24 hours were exposed to IL-1ß (10 ng/mL), IL-1ß plus PMA (10-7 mol/L), IL-1ß plus ET-1 (10-9 mol/L), or vehicle (-) for a further 24 hours. Data are means±SE of four samples. *P<.01.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we investigated whether ET-1 affected NO synthesis in VSMCs. Although ET-1 by itself had no effect on nitrite accumulation, it significantly inhibited IL-1ß–induced nitrite production. The inhibitory effect of ET-1 was reduced in the presence of the ET_A receptor antagonist BQ-485 and also in the PKC-depleted cells. These findings suggest that ET-1 acts on ET_A receptors and inhibits NO synthesis in IL-1ß–stimulated VSMCs at least partially through a PKC-dependent pathway.

The inhibitory effect was observed when ET-1 was given simultaneously or at least 2 hours before and up to 6 hours after IL-1ß treatment (data not shown). These findings strongly suggest a specific effect of ET-1 on iNOS expression and against an inhibitory effect of this molecule mediated through changes in IL-1ß receptor expression. Indeed, ET-1 reduced IL-1ß–evoked increases in iNOS mRNA and protein levels in VSMCs. Changes in mRNA transcription, stability, or both may account for the observed changes in mRNA levels. Nuclear run-off experiments will be necessary for direct assessment of transcription rates of the iNOS gene.

ET-1, a potent vasoconstrictor peptide secreted from endothelial cells, has been implicated in a number of human diseases, including hypertension and atherosclerosis.²⁰ ET-1 activities are mediated by binding to specific cell-surface receptors and coupled to phospholipase C–mediated hydrolysis and PKC activation. Two types of endothelin receptors, ET_A and ET_B, have been identified, cloned, and sequenced.^{21 22} The response of rat VSMCs to ET-1 is mediated by either the ET_A receptor or the ET_B receptor.¹⁷ The contraction and growth of VSMCs depend on the vascular action of ET-1 via the ET_A receptor.⁸ The vasodilator effect of ET-1 is induced by the binding of ET-1 to the ET_B receptor.²³ Recently, Beck et al²⁴ reported that ET-1 inhibited the formation of NO in rat glomerular mesangial cells elicited by cytokines via ET_A receptors, as observed in rat VSMCs in the present study. On the other hand, Nakahashi et al²⁵ reported that ET-1 affected neither basal nor IL-1ß–induced NO production by rat VSMCs. We speculate that the inhibitory effect of ET-1 on NO synthesis is mediated through a PKC-dependent pathway (Fig 6). Other PKC-activating agents such as angiotensin II^{4 26} and arginine vasopressin have also been shown to decrease cytokine-induced nitrite accumulation in rat VSMCs (unpublished data, 1996).

iNOS activity is induced in blood vessel wall and cultured VSMCs by endotoxins and cytokines.^{3 27} Joly et al²⁸ demonstrated that in vivo balloon injury induced NOS activity in rat carotid arteries, even in the absence of endothelium. Hansson et al²⁹ also reported that arterial smooth muscle cells in the neointima formed after deendothelializing balloon injury in the rat carotid artery expressed the cytokine-inducible isoform of NOS. Groves et al³⁰ showed that the NO donor molsidomine inhibited platelet adhesion and aggregation after angioplasty of the carotid artery in pigs. Several lines of evidence from both in vitro and in vivo studies have recently suggested a role of endogenous NO as an antiatherogenic autacoid³¹ ; NO inhibits aggregation of platelets,⁵ proliferation of VSMCs,⁶ production of cytokines by endothelial cells,³² expression of endothelial adhesion molecules,³³ adhesion of leukocytes to endothelial cells,⁷ and oxidation of low-density lipoprotein³⁴ and also causes vasorelaxation.³⁵ Thus, NO might be produced by the vascular tissue under various pathological conditions and inhibit de novo formation of intimal lesions.³¹ Recently, Cayatte et al³⁶ reported that chronic inhibition of NO synthesis with the NOS inhibitor N^G-nitro-L-arginine methyl ester accelerated neointimal formation in the ascending aorta of hypercholesterolemic rabbits, and Wang et al³⁷ reported that chronic administration of the NO precursor L-arginine prevented coronary atherogenesis in those rabbits. Furthermore, very recently, Wang et al³⁸ reported that ET_A and ET_B receptors were markedly upregulated in the rat carotid angioplasty model.

In conclusion, the present study revealed that ET-1 acts on ET_A receptors and inhibits NO synthesis in IL-1ß–stimulated VSMCs at least partially through a PKC-dependent pathway. These findings suggest that ET-1 may promote the initiation and progression of atherosclerosis by inhibiting endogenous NO production by VSMCs. However, further investigations are necessary to elucidate the mechanisms and conditions under which NO attenuates atherogenesis.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's minimum essential medium ET-1, -2, -3 = endothelin-1, -2, -3 ETA, ETB = endothelin type A, type B (receptor) FBS = fetal bovine serum IL-1ß = interleukin-1ß NO = nitric oxide NOS = nitric oxide synthase PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by the Ministry of Education, Science, Sports and Culture, Japan (No. 8670821).

Received May 15, 1996; first decision June 14, 1996; first decision August 14, 1996;

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