(Hypertension. 1995;25:744-747.)
© 1995 American Heart Association, Inc.
Articles |
Endothelin-1 Enhances Nitric Oxide–Induced Cytotoxicity in Vascular Smooth Muscle
From the Department of Geriatric Medicine, Osaka University Medical School, and Tsukuba Research Institute, Banyu Pharmaceutical Co Ltd, Ibaraki (M.Y.), Japan.
Correspondence to Keisuke Fukuo, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.
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Abstract |
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Abstract Prolonged incubation with 1 nmol/L interleukin-1 induced high levels of nitric oxide release and cytotoxicity in vascular smooth muscle cells. NG-Monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, inhibited interleukin-1–induced cytotoxicity at a concentration of 3 mmol/L. Furthermore, prolonged incubation with 0.1 mmol/L sodium nitroprusside, a nitric oxide donor, also induced cytotoxicity. On the other hand, endothelin-1 at concentrations from 10-10 to 10-7 mol/L induced a concentration-dependent enhancement of cytotoxicity induced by interleukin-1. However, endothelin-1 did not affect interleukin-1–induced nitric oxide production. Coculture study of vascular smooth muscle cells and endothelial cells without direct cell contact revealed that incubation for 72 hours with interleukin-1 induced high levels of nitric oxide release from cocultured vascular smooth muscle cells to the same degree as release from vascular smooth muscle cells alone. However, interleukin-1–induced cytotoxicity was more enhanced in cocultured vascular smooth muscle cells than in vascular smooth muscle cells alone. Furthermore, coincubation with 20 nmol/L BQ-485, an antagonist of one type of endothelin receptor (ETA), prevented the enhancement of interleukin-1–induced cytotoxicity in cocultured vascular smooth muscle cells. These findings suggest that endothelin-1 secreted from endothelial cells may enhance nitric oxide–induced cytotoxicity by means of the ETA receptor in vascular smooth muscle cells.
Key Words: nitric oxide • cytotoxins • muscle, smooth, vascular • endothelins • receptors, endothelin
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Nitric oxide (NO) is a physiological messenger formed by several cell types. NO produced by a Ca2+-dependent constitutive NO synthase is involved in the control of vascular tone, neuromodulation, and gut motility. The constitutive NO synthase system generates only small amounts of NO. In contrast, inflammatory stimuli, such as endotoxin and the cytokines, can induce a Ca2+-independent NO synthase that generates large amounts of NO in a variety of inflammatory cells, including macrophages and neutrophils, as well as in endothelial cells (EC) and vascular smooth muscle cells (VSMC).1 Because the atherosclerotic lesion consists of many inflammatory cells, cytokines and growth factors released from these cells may stimulate EC and VSMC in a paracrine manner, or themselves in an autocrine manner.2 Interleukin-1 (IL-1), a macrophage-derived cytokine, is known to stimulate the release of large amounts of NO from VSMC in vitro.3 Joly et al4 have also suggested that vascular smooth muscle contains a system that generates NO after balloon injury in vivo. Because NO has been implicated in tissue injury and cytotoxicity,5 6 we examined whether NO induces cytotoxicity in VSMC.
There is recent evidence that plasma levels of endogenous endothelin-1 (ET-1), a potent vasoconstrictor peptide secreted from EC, are increased in patients with symptomatic atherosclerosis,7 and ET-1 messenger RNA expression is elevated in human atherosclerotic lesions.8 Furthermore, Watanabe et al9 have recently demonstrated that endogenous ET-1 contributes to the extension of myocardial infarct size in rat. Therefore, we also examined whether ET-1 can modulate NO-induced cytotoxicity in VSMC.
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Methods |
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Materials
Human recombinant IL-1 (IL-1ß) was donated by Otsuka Pharmaceutical Co. NG-Monomethyl-L-arginine (L-NMMA) and sodium nitroprusside (SNP) were purchased from Sigma Chemical Co. ET-1 was from Peptide Institute Inc. [3H]Leucine (5 mCi/mL) was obtained from Amersham International. BQ-485, a selective antagonist of one type of endothelin receptor (ETA), was identified as described previously.10
Cell Culture
VSMC were isolated from Wistar rat aortas as described
previously.11 Bovine aortic EC were isolated following the
method of Jaffe et al.12 For the coculture system, EC
(passages 4 through 8) were grown to confluence on the collagen-coated
microporous membrane of transwells (12-mm diameter, 3.0-µm pore size;
Costar). After the VSMC reached confluence, they were preincubated with
serum-free Dulbecco's modified Eagle medium (DMEM) containing 0.1%
bovine serum albumin so they would become quiescent. The coculture
system was then prepared by placing the transwells into the wells of
the culture plate, and both EC and VSMC were cultured in DMEM
containing 0.1% bovine serum albumin in the presence or absence of 1
nmol/L IL-1 or other compounds. In this coculture system, the culture
medium was shared by both types of cells. Therefore, humoral
interchange was allowed between them without direct cell contact.
Nitrite Assay
We measured the level of nitrite in the medium as a reflection
of NO production as described previously.13 The nitrite
level in cell-free supernatant was determined using Griess reagent
consisting of 1% sulfanilamide, 0.1%
naphthylethylene-diamine-dihydrochloride, and 2%
H3PO4. Nitrite concentration, proportional to
the optical density reached at 540 nm (OD540), was
determined by use of a microtiter plate reader (model 450, Bio-Rad
Laboratories) with reference to NaNO2 as a standard.
Cytotoxicity Assay
Quiescent VSMC cultured in 24-well plates or VSMC cocultured in
12-well plates with EC were incubated for the indicated times with 1
nmol/L IL-1 or other compounds. After the incubation, the medium was
collected and stored at -20°C until assay. Cytotoxicity was
quantitated by measurement of release of the soluble cytoplasmic enzyme
lactate dehydrogenase (LDH) into culture supernatant. LDH activity in
cell-free supernatant was measured using a commercial kit (Sigma
Chemical Co). Total LDH activity (136.2±9.4 U/L; n=8) was determined
from the supernatant of a sample of VSMC incubated with 0.1% Triton
X-100 for 30 minutes. Cells were then labeled with
[3H]leucine (2 µCi/mL) for a further 4 hours. After the
labeling, cells were washed three times with cold PBS and treated with
5% trichloroacetic acid and ethanol-ethylether (3:1, vol/vol). The
residues in the wells were solubilized in 0.3N NaOH, and the
radioactivity of aliquots of the solution was measured after the
neutralization of pH.
Statistical Analysis
Statistical analysis was performed by one-way ANOVA. Results
are expressed as mean±SEM. A value of P<.05 was considered
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Fig 1 shows the kinetics of NO production and LDH release from VSMC after exposure to IL-1 (1 nmol/L). IL-1 induced a time-dependent and high level of NO release from VSMC (Fig 1A). On the other hand, prolonged incubation for 72 hours with IL-1 induced significant stimulation of LDH release from VSMC, whereas short-term incubation for 24 hours with IL-1 did not induce LDH release. However, coincubation with 3 mmol/L L-NMMA, an inhibitor of NO synthesis, significantly inhibited IL-1–induced stimulation of both NO and LDH release at a concentration higher than that of L-arginine in the medium (0.4 mmol/L). Furthermore, prolonged incubation for 72 hours with SNP, an NO donor, also induced LDH release from VSMC.
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Next, we examined whether ET-1, a strong vasoconstrictive peptide secreted from EC, modulates NO-induced cytotoxicity in VSMC. As shown in Fig 2A, ET-1 affected neither basal nor IL-1–induced NO release from VSMC. However, although ET-1 itself did not affect LDH release from VSMC, coincubation with ET-1 induced a concentration-dependent enhancement of LDH release from VSMC incubated for 72 hours with IL-1 (Fig 2B).
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We then examined whether ET-1 enhances cytotoxicity in response to SNP in VSMC. Although ET-1 did not affect SNP-induced NO release (Fig 3A,) ET-1 enhanced both LDH release and inhibition of [3H]leucine incorporation induced by SNP (the absolute value of [3H]leucine incorporation in control culture was 41 627±3038 cpm per well) (Fig 3B). In contrast, angiotensin II (10-8 mol/L) did not affect IL-1–induced nitrite production (Fig 3A), LDH release, or inhibition of [3H]leucine incorporation (Fig 3B).
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Next, we examined whether prolonged incubation with IL-1 can induce cytotoxicity in VSMC when VSMC are cocultured with EC. As shown in Fig 4A, prolonged incubation for 72 hours with IL-1 induced high levels of NO release from VSMC cocultured with EC to the same degree as in VSMC alone. It also induced significant inhibition of [3H]leucine incorporation in cocultured VSMC (the absolute value in cocultured VSMC was 58 225±3319 cpm per well). However, the degree of inhibition of [3H]leucine incorporation was more enhanced in cocultured VSMC than in VSMC alone. On the other hand, coincubation with 20 nmol/L BQ-485, a selective ETA receptor antagonist, prevented the enhancement of IL-1–induced inhibition of [3H]leucine incorporation in cocultured VSMC. Furthermore, the addition of exogenous ET-1 (10-7 mol/L) to cocultured VSMC treated with BQ-485 restored inhibition of [3H]leucine incorporation induced by IL-1 (Fig 4B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, prolonged incubation with IL-1 induced both high levels of NO production and cytotoxicity in VSMC. L-NMMA, an inhibitor of NO synthesis, inhibited both NO production and cytotoxicity induced by IL-1. Furthermore, SNP, an NO donor, also induced cytotoxicity in VSMC, suggesting that NO mediates IL-1–induced cytotoxicity in VSMC. NO has been implicated in tisssue injury because of its capacity to interact with oxygen-derived free radicals to produce toxic substances such as the peroxynitrite anion.5 It also binds and inactivates iron-sulfur–containing enzymes that are important in the regulation of cell function and viability.6 Recently, Ellman et al14 reported a similar result: that NO may mediate IL-1–induced cytotoxicity in rat ovary. Patel et al15 have also reported that inhibition of NO limits infarct size in the in situ rabbit heart after sustained coronary artery occlusion and reperfusion. However, Fung et al16 have reported the opposite result: that an NO inhibitor exacerbates necrosis in the rabbit heart during ischemia-reperfusion. Taken together, these findings suggest that NO may participate in the mechanism of tissue injury in the cardiovascular system.
ET-1, a potent vasoconstrictor peptide secreted from EC, is implicated in a number of human diseases, including hypertension and atherosclerosis.17 Furthermore, ET-1 activities are mediated by binding to specific cell surface receptors. Two types of ET receptors, ETA and ETB, have been identified, cloned, and sequenced.18 19 Recently, Gellai et al20 reported that treatment with a selective ETA receptor antagonist reverses severe renal damage in ischemia-induced acute renal failure of the rat. Furthermore, Watanabe et al9 have recently demonstrated that endogenous ET contributes to the extension of myocardial infarct size in rats. Although a strong vasoconstrictive effect is thought to be most responsible for ET-induced tissue damage, there is evidence that ET-1 may directly potentiate the cellular damage induced by ischemia in cultured rat myocytes.21
In this study, ET-1 enhanced not only IL-1–induced but also SNP-induced cytotoxicity, suggesting that ET-1 enhances NO-induced cytotoxicity in VSMC. On the other hand, the degree of NO-induced cytotoxicity was more enhanced in cocultured VSMC with EC than in VSMC alone. However, BQ-485 prevented the enhancement of NO-induced cytotoxicity in cocultured VSMC. Furthermore, the addition of exogenous ET-1 (10-7 mol/L) to cocultured VSMC treated with BQ-485 restored IL-1–induced cytotoxicity. Taken together, these findings suggest that endogenous ET-1 enhances NO-induced cytotoxicity by means of the ETA receptor in VSMC. ET-1–induced enhancement might be ET-1 specific, because angiotensin II did not enhance NO-induced cytotoxicity in VSMC. Furthermore, our preliminary experiments suggest that the enhancement of NO-induced cytotoxicity is prevented by coincubation with superoxide dismutase and catalase in VSMC (T.N. et al, unpublished data, 1994). Thus, oxygen-derived free radicals and oxidants might be involved in the mechanism of the enhancement of cytotoxicity induced by ET-1 in VSMC. However, further studies are necessary to clarify this point.
In conclusion, our results indicate that endogenous ET-1 may enhance cytotoxicity induced by high levels of NO by means of the ETA receptor in VSMC.
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Acknowledgments |
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This work was supported by a grant from Uehara Memorial Foundation. We thank Taeko Kaimoto for her excellent technical assistance.
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References |
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