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(Hypertension. 1995;25:711-714.)
© 1995 American Heart Association, Inc.

Articles

cGMP Upregulates Nitric Oxide Synthase Expression in Vascular Smooth Muscle Cells

Takuo Inoue; Keisuke Fukuo; Takeshi Nakahashi; Shigeki Hata; Shigeto Morimoto; Toshio Ogihara

From the Department of Geriatric Medicine, Osaka (Japan) University Medical School.

Correspondence to Keisuke Fukuo, MD, Department of Geriatric Medicine, Osaka University Medical School, Suita, Osaka 565, Japan.

	Abstract

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Abstract 8-Bromo-guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), an analogue of cyclic guanosine monophosphate (cGMP), induced a time- and dose-dependent enhancement of interleukin-1–induced nitric oxide production in vascular smooth muscle cells. Human atrial natriuretic polypeptide, which stimulates cGMP accumulation in vascular smooth muscle cells, also enhanced interleukin-1–induced nitric oxide release at a concentration of 100 nmol/L. In contrast, coincubation with 10 µmol/L methylene blue, an inhibitor of soluble guanylate cyclase, inhibited interleukin-1–induced nitric oxide release from vascular smooth muscle cells. Furthermore, coincubation with 8-Br-cGMP also enhanced the interleukin-1–induced increase in inducible nitric oxide synthase messenger RNA in vascular smooth muscle cells. However, the enhancement of nitric oxide production induced by 8-Br-cGMP was significantly prevented by coincubation with neutralizing antibody against tumor necrosis factor–. Furthermore, 8-Br-cGMP enhanced the interleukin-1–induced increase in tumor necrosis factor– messenger RNA level in vascular smooth muscle cells. These findings indicate that cGMP may upregulate inducible nitric oxide synthase gene expression through the stimulation of tumor necrosis factor– production in vascular smooth muscle cells. Thus, there may be a positive feedback mechanism between nitric oxide and the cGMP system in vascular smooth muscle cells.

Key Words: nitric oxide • guanosine cyclic monophosphate • muscle, smooth, vascular • tumor necrosis factor • interleukin-1

	Introduction

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Nitric oxide (NO) is an unstable but multifunctional molecule that mediates many physiological processes.^{1 2} At least two distinct forms of NO synthase (NOS) have been cloned.³ One is constitutively expressed and is present in vascular endothelial cells and the brain. The other (iNOS) is inducibly expressed and has been identified in cytokine-treated macrophages, endothelial cells, and vascular smooth muscle cells (VSMC).^{4 5 6} Macrophage-type iNOS is activated by bacterial lipopolysaccharide and several cytokines such as interleukin-1 (IL-1), tumor necrosis factor– (TNF-), and interferon gamma. NO, in turn, activates soluble guanylate cyclase, generating cyclic guanosine monophosphate (cGMP) that mediates relaxation in VSMC. There is recent evidence that cGMP upregulates TNF- synthesis in rat peritoneal macrophages.⁷ Furthermore, Eigler et al⁸ have recently shown that NO-releasing agents enhance cytokine-induced TNF- synthesis in human mononuclear cells. Therefore, we examined the possibility that cGMP may modulate iNOS expression through the stimulation of TNF- synthesis in VSMC.

	Methods

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Materials
Human recombinant IL-1ß was provided by Otsuka Pharmaceutical Co Inc. N^G-Monomethyl-L-arginine (L-NMMA), 8-bromo-guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), and methylene blue were from Sigma Chemical Co. Human atrial natriuretic polypeptide (ANP) was purchased from Peptide Institute. Human recombinant TNF- and a human TNF- complementary DNA (cDNA) probe were kindly provided by Dainippon Pharmaceutical Co, Ltd. Mouse monoclonal antibody against human TNF- was purchased from Promega Co. The cDNA probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from the American Type Culture Collection (ATCC No. 57091).

Cells
VSMC were isolated from Wistar rat aorta as described previously.⁹ VSMC (passages 4 through 9) were grown to confluence on 24-well plates or 10-cm dishes with Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum. Cells were then preincubated for 48 hours with serum-free DMEM containing bovine serum albumin to become quiescent before the addition of IL-1 or other compounds.

Nitrite Assay
We measured the nitrite level in cell-free supernatant as a reflection of NO production by using Griess reagent, as described previously.¹⁰

Analysis of RNA
Total RNA extraction and Northern blot analysis were performed as described previously.¹¹ The iNOS cDNA probe for Northern blot analysis was prepared by polymerase chain reaction, as recently reported.¹² The expected size of the polymerase chain reaction product (577 bp) was ligated into the pGEM plasmid vector (Promega) by the thymine/adenine (T/A) cloning method. An insert of BamHI-EcoRI restriction fragment of iNOS plasmids was used for Northern blot analysis. The cDNA probes for mouse iNOS, human TNF-, and human GAPDH were labeled with [³²P]deoxycytidine triphosphate (111 TBq/mmol) by the random-primed labeling method. Hybridization with a GAPDH cDNA probe was used to monitor uniform loading of RNA on Northern blots.

Statistical Analysis
Statistical analysis was performed by one-way ANOVA. Results are expressed as mean±SEM. A value of P<.05 was considered significant.

	Results

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IL-1 induced a time-dependent and high level of NO release from VSMC. L-NMMA, an inhibitor of NO synthesis, inhibited NO release from VSMC induced by IL-1 (Fig 1A). Northern blot analysis revealed that IL-1 induced a time-dependent increase in iNOS messenger RNA (mRNA) level in VSMC. Maximal increase was observed at 12 hours after the stimulation (Fig 1B).

View larger version (21K): [in this window] [in a new window]   Figure 1. Line graph (A) shows kinetics of nitric oxide release, and Northern blot analysis (B) shows inducible nitric oxide synthase (iNOS) messenger RNA expression after stimulation of vascular smooth muscle cells with interleukin-1 (IL-1). Quiescent cells were incubated with IL-1 (1 nmol/L) in the presence or absence of NG-monomethyl-L-arginine (L-NMMA) (3 mmol/L) for the times indicated. Nitrite concentration and iNOS messenger RNA level were determined as described in text. Values are mean±SEM of four individual experiments containing three replications in each experiment. *P<.05, significantly different from control; **P<.05, significantly different from cells treated with IL-1 alone.

Although 8-Br-cGMP, a cGMP analogue, induced a weak stimulation of NO release from VSMC at a concentration of 1 mmol/L, it significantly induced a time-dependent enhancement of NO release induced by IL-1 (Fig 2A). Furthermore, 8-Br-cGMP also induced a dose-dependent enhancement of NO release from VSMC incubated for 48 hours with IL-1 (Fig 2B). In addition, IL-1 (1 nmol/L) actually stimulated cGMP accumulation (pmol/mg protein) in VSMC (1.8±0.2 at 0 hours, 3.5±0.4 at 1 hour, 17.2±3.7 at 3 hours, 71.5±10.8 at 6 hours, and 118.6±15.5 at 24 hours) in the presence of 0.5 mmol/L 3-isobutyl-1-methylxanthine (results of two different experiments; containing two replicates in each experiment; all data except those from 0 hours were significantly different from control).

View larger version (17K): [in this window] [in a new window]   Figure 2. Line graphs show time- and dose-dependent effects of 8-bromo-guanosine 3':5'-cyclic monophosphate (8Br-cGMP) on nitric oxide production in vascular smooth muscle cells (VSMC). For the time-course study (A), quiescent VSMC were incubated for the times indicated with 8Br-cGMP (2 mmol/L), interleukin-1 (IL-1; 1 nmol/L), or both. For the dose-response study (B), quiescent VSMC were incubated for 48 hours with 8Br-cGMP at the concentrations indicated in the presence or absence of IL-1 (1 nmol/L). Nitrite concentration in the medium was determined as described in text. Values are mean±SEM of four different experiments containing two replications in each experiment. *P<.05, significantly different from control; **P<.05, significantly different from cells treated with IL-1 alone.

As shown in Fig 3, coincubation with 10 µmol/L methylene blue, an inhibitor of soluble guanylate cyclase, partially inhibited IL-1–induced NO release from VSMC. However, ANP, which stimulates cGMP accumulation in VSMC through the activation of guanylate cyclase–linked ANP receptors, significantly enhanced IL-1–induced NO release from VSMC at a concentration of 100 nmol/L (Fig 3).

View larger version (34K): [in this window] [in a new window]   Figure 3. Bar graph shows effects of 8-bromo-guanosine 3':5'-cyclic monophosphate (8Br-cGMP), methylene blue (MB), atrial natriuretic peptide (ANP), and tumor necrosis factor– (TNF-) on interleukin-1 (IL-1)–induced nitric oxide production in vascular smooth muscle cells. Quiescent vascular smooth muscle cells were incubated for 48 hours with serum-free medium containing 8Br-cGMP (2 mmol/L), IL-1 (1 nmol/L), MB (10 µmol/L), ANP (100 nmol/L), and TNF- (10 ng/mL) in the presence or absence of 10 µg/mL anti–TNF- IgG or control IgG. Nitrite concentration in the medium was determined as described in text. Values are mean±SEM of four different experiments containing two replications in each experiment. *P<.05, significantly different from control; **P<.05, significantly different from cells treated with IL-1 alone; +P<.05, significantly different from cells treated with IL-1+TNF-; ++P<.05, significantly different from cells treated with IL-1 plus 8Br-cGMP; and NS, no significant difference.

To examine whether TNF- is involved in the enhancement of NO release induced by 8-Br-cGMP, we used monoclonal antibody against TNF- to neutralize its effect. Although 10 ng/mL TNF- significantly enhanced IL-1–induced NO release from VSMC, monoclonal antibody against TNF- neutralized its enhancement. Furthermore, anti–TNF- IgG significantly inhibited the 8-Br-cGMP–induced enhancement of NO release from VSMC incubated with IL-1, whereas nonimmune IgG had no effect (there was no significant difference between VSMC with IL-1 plus 8-Br-cGMP and VSMC with IL-1 plus 8-Br-cGMP plus control IgG) (Fig 3).

We then examined whether cGMP can modulate iNOS gene expression in VSMC. Although incubation for 6 hours with 2 mmol/L 8-Br-cGMP alone induced a small increase in iNOS mRNA level, it significantly upregulated iNOS mRNA expression induced by IL-1 in VSMC. Furthermore, 8-Br-cGMP also enhanced the increase in TNF- mRNA level induced by IL-1. However, no significant changes of GAPDH mRNA levels were observed after treatment with IL-1 or 8-Br-cGMP (Fig 4).

View larger version (51K): [in this window] [in a new window]   Figure 4. Northern blot analysis showing effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) on interleukin-1 (IL-1)–induced inducible nitric oxide synthase (iNOS) and tumor necrosis factor– (TNF-) messenger RNA in vascular smooth muscle cells. Quiescent vascular smooth muscle cells were incubated for 6 hours with IL-1 (1 nmol/L) or 8Br-cGMP (2 mmol/L). Total RNA extraction and Northern blot analysis were carried out as described in text. Signals for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA for each lane are shown as control at the bottom.

	Discussion

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The present study demonstrated that cGMP upregulates IL-1–induced iNOS expression in VSMC. This conclusion is based on the following results: (1) 8-Br-cGMP, a membrane-permeable cGMP derivative, induced a time- and dose-dependent enhancement of IL-1–induced stimulation of NO release from VSMC; (2) coincubation with methylene blue, an inhibitor of soluble guanylate cyclase, inhibited IL-1–induced NO release from VSMC; (3) coincubation with ANP, which binds the ANP_B receptor, activates a membrane-type guanylate cyclase, and thus results in the accumulation of intracellular cGMP in VSMC,¹³ enhanced IL-1–induced NO production in VSMC; and (4) 8-Br-cGMP enhanced the increase in iNOS mRNA level induced by IL-1 in VSMC.

Although the pathophysiological roles of NO released from VSMC have not been defined, we have recently demonstrated that prolonged incubation of VSMC for 72 hours with IL-1 induced cytotoxicity through production of high levels of NO in VSMC themselves. However, 3 mmol/L L-NMMA inhibited both NO production and cytotoxicity induced by IL-1 in VSMC, suggesting that NO released from VSMC is involved in the mechanism of vascular remodeling in atherosclerotic plaques.¹⁴

TNF was originally defined by its tumoricidal activity in vivo and cytotoxic activity in vitro.¹⁵ Several studies have shown that a wide range of biological activities of TNF and IL-1 overlap and are indistinguishable.¹⁶ There is recent evidence that TNF- synthesis in macrophages is upregulated by cGMP and downregulated by cAMP.⁷ Furthermore, Eigler et al⁸ have recently reported that NO-releasing agents enhance IL-1–induced TNF synthesis in human mononuclear cells. In this study, we have demonstrated that neutralization of the biological activity of TNF- with a specific monoclonal antibody inhibited 8-Br-cGMP–induced enhancement of NO production from VSMC. Furthermore, coincubation with 8-Br-cGMP enhanced TNF- mRNA expression induced by IL-1 in VSMC.

These findings suggest that cGMP upregulates IL-1–induced NO production by stimulating the formation of TNF- in VSMC. Thus, cGMP may function as a positive feedback modulator of NO synthesis by enhancing TNF- production in VSMC.

	Acknowledgments

 
This work was supported by a grant from Uehara Memorial Foundation. We thank Taeko Kaimoto for her excellent technical assistance.

	References

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