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(Hypertension. 1996;27:1240-1244.)
© 1996 American Heart Association, Inc.

Articles

Adrenomedullin Increases Inducible Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Stimulated With Interleukin-1

Uichi Ikeda; Toshiko Kanbe; Kazuyuki Shimada

From 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 vyg02111@niftyserve.or.jp.

	Abstract

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Abstract We investigated the effects of adrenomedullin on nitric oxide synthesis by measuring the production of nitrite, a stable metabolite of nitric oxide, in cultured rat vascular smooth muscle cells. Incubation of cultures with interleukin-1ß (10 ng/mL) for 24 hours caused a significant increase in nitrite generation. The interleukin-1ß–induced nitrite production by vascular smooth muscle cells was significantly increased by adrenomedullin in a dose-dependent manner (10^-10 to 10^-6 mol/L). This effect of adrenomedullin was significantly inhibited in the presence of N^G-monomethyl-L-arginine. The adrenomedullin-induced nitrite production by interleukin-1ß–stimulated cells was accompanied by increased inducible nitric oxide synthase mRNA accumulation. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, interleukin-1ß–induced nitrite accumulation was further increased, but the effect of adrenomedullin was not additive or synergistic. Adrenomedullin dose dependently increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that adrenomedullin augments nitric oxide synthesis in interleukin-1ß–stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

Key Words: adrenomedullin • interleukin-1 • nitric oxide • cAMP • muscle, smooth, vascular

	Introduction

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Nitric oxide (NO), the extensively characterized endothelium-derived relaxing factor, is a short-lived free radical. NO is synthesized from L-arginine by three isoenzymes expressed either constitutively (neuronal, type I cNOS; endothelial, type III cNOS) or after stimulation by cytokines (inducible, type II iNOS).^{1 2} Inducible NO synthase has been identified in endotoxin- and cytokine-treated neutrophils, hepatocytes, endothelial cells, and myocardium.^{1 3} Inducible NO synthase activity is also induced in aortic rings and cultured vascular smooth muscles by cytokines and endotoxins.^{4 5} It has also been demonstrated that in vivo balloon injury induces NO synthase activity in rat carotid arteries.⁶ Therefore, NO synthase induction in vascular smooth muscle cells (VSMCs) may play a role in vascular contractility and local vascular injury associated with hypertension or atherosclerosis.

Adrenomedullin, a potent endogenous vasodilating peptide, has recently been isolated from the acid extract of human pheochromocytoma.⁷ This peptide, consisting of 52 amino acids, has one intracellular disulfide bond and shows approximately 20% homology with calcitonin gene–related peptide. Adrenomedullin injected intravenously causes a potent and long-lasting hypotensive effect in anesthetized rats.⁸ It has been shown that VSMCs express adrenomedullin-specific receptors. A binding study with ¹²⁵I-adrenomedullin revealed the presence of a single class of high-affinity (K_d=1.3x10^-8 mol/L) binding sites for adrenomedullin in rat VSMCs.⁹ Recently, Ishizaka et al¹⁰ and Eguchi et al⁹ demonstrated that adrenomedullin stimulates cAMP formation in rat VSMCs. Adrenomedullin thus acts directly on vascular smooth muscle and modulates vascular contractility and metabolism. However, no reports have concerned the effects of adrenomedullin on the production of NO, another modulator of vascular function, by vascular smooth muscle. Therefore, in this study, we investigated the effects of adrenomedullin on NO synthesis in cultured rat VSMCs.

	Methods

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Cell Culture
Primary cultures of VSMCs were obtained from the mediae of thoracic aortas of Sprague-Dawley rats (200 to 250 g), as described previously.¹¹ The cells were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, 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 experiment was performed with cultured cells at passage levels of 5 to 10. Cells (3x10⁴/mL) were plated onto 24-well or 100-mm culture dishes in DMEM, supplemented as described above, and allowed to grow to subconfluence for 24 to 48 hours, after which they were preincubated in DMEM containing 0.5% fetal bovine serum and supplemented with insulin (5 µg/mL) and transferrin (5 µg/mL) for 24 hours and then used for the experiments described below.

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

Nitrite Measurements
NO production by the cultured cells was determined by measurement of the nitrite contents of the culture media. VSMCs plated in 24-well dishes were incubated in DMEM containing 0.5% fetal bovine serum at 37°C. The nitrite contents of culture media were determined by mixing 500 µL medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid).¹² The absorbance at 550 nm was measured, and the nitrite concentration was 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 and used for protein assay (Bio-Rad assay kit) with bovine serum albumin as a standard. Nitrite levels were corrected by protein measurement, and data are shown as nanomoles per milligram protein.

Northern Blot Analysis
Total RNA was extracted from VSMCs plated in 100-mm culture dishes by the guanidinium isothiocyanate/cesium chloride procedure,¹³ and 30-µg aliquots were subjected to electrophoresis on 1% agarose gels. After electrophoretic separation, RNA was transferred onto nylon filters, which were then hybridized with a random-primed ³²P-labeled mouse macrophage inducible NO synthase cDNA probe for 24 hours,¹⁴ and washed twice with an aqueous solution of 150 mmol/L NaCl, 15 mmol/L sodium citrate, and 0.1% sodium dodecyl sulfate at 65°C. The filters were exposed to Kodak XAR-5 film for 1 to 2 days at -70°C with the use of one intensifying screen.

cAMP Measurements
For determination of intracellular cAMP levels, 0.5 mmol/L isobutylmethylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor, was added to each well 30 minutes before the addition of adrenomedullin to prevent breakdown of accumulated cAMP. After incubation with adrenomedullin for 60 minutes, cells were immediately immersed in 0.2 mL of 0.1N HCl to stop the reaction. Cells were then collected in glass tubes with a rubber policeman, boiled for 5 minutes, and centrifuged at 2500g for 15 minutes at room temperature. The supernatants were decanted, and after 0.05 mL of 50 mmol/L sodium acetate was added to each tube, cells were kept at -70°C until assay for cAMP contents. The pellets were dissolved in 0.2 mL of 1% sodium dodecyl sulfate and kept at 4°C until assayed for protein. Intracellular cAMP contents were measured with a commercial enzyme immunoassay kit using the manufacturer's high-sensitivity acetylation protocol (Amersham Life Science). The lower limit of detection was 2 fmol per well. The values were normalized to protein content of each well.

Statistical Analysis
Data are expressed as mean±SD of four samples, which represented at least three separate experiments. Differences were analyzed by one-way ANOVA combined with Scheffé's test, and values of P<.05 were considered to be statistically significant.

Materials
Rat adrenomedullin was purchased from Peptide Institute Inc. Human recombinant interleukin-1ß (specific activity, 2x10⁷ U/mg) was a gift from Otsuka Pharmacy. Mouse inducible NO synthase cDNA was a gift from Dr Y. Kawahara (Kobe [Japan] University School of Medicine). IBMX and N^G-monomethyl-L-arginine (L-NMMA) were from Sigma Chemical Co. All other chemicals used were of the highest grade commercially available.

	Results

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Effects of Adrenomedullin on Nitrite Production
First, we investigated the effects of interleukin-1ß on nitrite production by VSMCs. As shown in Fig 1, addition of interleukin-1ß (10 ng/mL) stimulated nitrite production by VSMCs in a time-dependent manner. This interleukin-1ß–stimulated nitrite accumulation was significantly increased by simultaneous treatment of the cells with adrenomedullin (10^-7 mol/L). After a 24-hour incubation, the level of interleukin-1ß–stimulated nitrite accumulation in the presence of adrenomedullin was about twofold that in the absence of adrenomedullin.

View larger version (14K): [in this window] [in a new window]   Figure 1. Time course of nitrite accumulation stimulated by interleukin-1ß in the presence or absence of adrenomedullin in vascular smooth muscle cells. Cells were unstimulated () or stimulated with 10 ng/mL interleukin-1ß in the presence () or absence () of 10-7 mol/L adrenomedullin for various times as indicated. Nitrite accumulation in the culture medium was measured as described in "Methods," and values were normalized to the protein content per dish. Points and bars represent mean±SD (n=4). *P<.05, **P<.01 compared with interleukin-1ß–stimulated cells.

Fig 2 shows the dose-response relationship of the effect of adrenomedullin. Adrenomedullin increased interleukin-1ß–stimulated nitrite production by VSMCs in a dose-dependent manner (10^-10 to 10^-6 mol/L). Adrenomedullin by itself did not affect the basal level of nitrite production.

View larger version (17K): [in this window] [in a new window]   Figure 2. Dose-dependent effect of adrenomedullin on nitrite accumulation. Vascular smooth muscle cells were incubated for 24 hours with (solid bars) or without (hatched bars) 10 ng/mL interleukin-1ß in the presence of various concentrations of adrenomedullin (10-10 to 10-6 mol/L) as indicated. Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per dish. Data represent mean±SD (n=4). *P<.05, **P<.01 compared with control cells without adrenomedullin.

As shown in Fig 3, in the presence of the NO synthesis inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mmol/L), the effects of adrenomedullin and interleukin-1ß were abolished.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effects of NG-monomethyl-L-arginine (L-NMMA) on interleukin-1ß– and adrenomedullin-induced nitrite accumulation in vascular smooth muscle cells. Cells were incubated for 24 hours with 10-7 mol/L adrenomedullin (AM), 10 ng/mL interleukin-1ß (IL-1ß), and/or 1 mmol/L L-NMMA. Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per dish. Data represent mean±SD (n=4). *P<.01 compared with control cells (-).

Temporal Analysis of Stimulatory Action of Adrenomedullin
As shown in Fig 4, the addition of adrenomedullin either 3 or 6 hours after treatment of the cells with interleukin-1ß still increased nitrite production, although the stimulatory effect was decreased. However, no significant stimulatory effect was observed when adrenomedullin was added 12 hours after interleukin-1ß treatment.

View larger version (12K): [in this window] [in a new window]   Figure 4. Temporal analysis of the stimulatory action of adrenomedullin. Vascular smooth muscle cells were stimulated for 24 hours with 10 ng/mL interleukin-1ß. Adrenomedullin (10-7 mol/L) was added either simultaneously or 3, 6, or 12 hours after interleukin-1ß stimulation. Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per dish. *P<.05, **P<.01 compared with control cells (-).

Effects of Adrenomedullin on Inducible NO Synthase mRNA Levels
Since the temporal analysis described above strongly suggested that adrenomedullin increased interleukin-1ß–stimulated NO production at the level of inducible NO synthase expression, we examined whether adrenomedullin actually induced increases in inducible NO synthase mRNA levels in VSMCs. As shown in Fig 5, unstimulated cells did not express inducible NO synthase mRNA. Incubation with interleukin-1ß (10 ng/mL) for 12 to 24 hours resulted in induction of inducible NO synthase mRNA levels, and the levels were further increased in the presence of adrenomedullin (10^-7 mol/L).

View larger version (68K): [in this window] [in a new window]   Figure 5. Expression of inducible NO synthase (iNOS) mRNA in vascular smooth muscle cells. Cells were incubated with 10 ng/mL interleukin-1ß (IL-1) for 12 or 24 hours with or without 10-7 mol/L adrenomedullin (AM). Total RNA was size-fractionated by electrophoresis and transferred onto nylon filters. Filters were hybridized with 32P-labeled mouse macrophage inducible NO synthase (top) and 18S rRNA (bottom) cDNA probes. (-) indicates control sample not exposed to interleukin-1ß and adrenomedullin. Two independent experiments yielded identical results.

Involvement of cAMP in the Action of Adrenomedullin
We then investigated the mechanism of the stimulatory effect of adrenomedullin on interleukin-1ß–induced NO production. It has been shown that a cAMP-dependent pathway is involved in cytokine-induced NO production by VSMCs.^{14 15 16} As shown in Fig 6, in the presence of the phosphodiesterase inhibitor IBMX, interleukin-1ß–induced nitrite accumulation was significantly increased, but the effect of adrenomedullin was not additive or synergistic, suggesting that the effect of adrenomedullin is mediated by a cAMP-dependent pathway.

View larger version (24K): [in this window] [in a new window]   Figure 6. Effects of isobutylmethylxanthine on nitrite accumulation in vascular smooth muscle cells. Cells were incubated for 24 hours with 10 ng/mL interleukin-1ß (IL-1ß) and/or 10-7 mol/L adrenomedullin (AM) in the presence (solid bars) or absence (hatched bars) of 0.5 mmol/L isobutylmethylxanthine. Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per dish. Data represent mean±SD (n=4). *P<.05.

We thus actually measured intracellular cAMP levels of VSMCs by adrenomedullin. As shown in Fig 7, addition of adrenomedullin for 1 hour dose dependently (10^-9 to 10^-6 mol/L) increased intracellular cAMP levels of VSMCs.

View larger version (16K): [in this window] [in a new window]   Figure 7. Effects of adrenomedullin on intracellular cAMP levels of vascular smooth muscle cells. Cells were incubated for 1 hour with 10-9 to 10-6 mol/L adrenomedullin in the presence of 0.5 mmol/L isobutylmethylxanthine added 30 minutes before addition of adrenomedullin. Intracellular cAMP contents were measured as described in "Methods," and values were normalized to the protein content per dish. Data represent mean±SD (n=4). *P<.05, **P<.01 compared with control cells (-).

	Discussion

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Adrenomedullin is a peptide recently isolated from pheochromocytoma that shows vasorelaxant and long-lasting hypotensive effects.⁸ This peptide has been shown to be present not only in human adrenal medulla but also in lung, cardiac ventricle, kidney, and circulating blood.^{17 18} In the present study, we investigated whether adrenomedullin modulates NO synthesis in VSMCs. Although adrenomedullin by itself showed no effect on NO production, it augmented interleukin-1ß–induced NO production by VSMCs in a dose-dependent manner. The stimulatory effect of adrenomedullin was significantly reduced when added several hours after the addition of interleukin-1ß, suggesting that the augmentation of nitrite production by adrenomedullin is due to induction of inducible NO synthase. Indeed, we observed that adrenomedullin increased interleukin-1ß–induced inducible NO synthase mRNA levels in VSMCs.

We obtained three pieces of evidence for a causal link between production of cAMP and augmentation of NO synthesis by adrenomedullin in VSMCs. First, adrenomedullin augmented interleukin-1ß–induced NO production, and this effect was accompanied by an increase in the cellular levels of cAMP. Second, the effect of adrenomedullin on interleukin-1ß–induced NO production was not additive or synergistic in the presence of IBMX. Third, the cAMP analogue dibutyl-cAMP increased interleukin-1ß–induced NO production by cultured rat VSMCs (data not shown), as reported previously by other investigators.^{14 15 16} These results suggest that adrenomedullin augments interleukin-1ß–induced NO production in VSMCs, at least partially through a cAMP-dependent process.

The data presented here do not address the molecular mechanism by which adrenomedullin or cAMP alters the inducible NO synthase mRNA levels in interleukin-1ß–stimulated VSMCs. Changes in the transcription and/or mRNA stability may account for the observed changes in mRNA levels. From the potent inhibitory action of actinomycin D (data not shown) and the lag time of several hours before the onset of inducible NO synthase activity (Fig 1), transcriptional activation of inducible NO synthase expression seems a likely explanation for our observations. However, nuclear run-on experiments will be necessary for direct assessment of transcription rates of the inducible NO synthase gene.

In the present study, adrenomedullin significantly increased interleukin-1ß–stimulated NO production at 10^-8 to 10^-7 mol/L. According to Sakata et al¹⁹ and Ichiki et al,¹⁸ mean plasma concentrations of adrenomedullin in rats and humans are 3.6±0.3x10^-9 mol/L (mean±SD) and 3.3±0.4x10^-9 mol/L, respectively. Under the current experimental conditions, supraphysiological doses of adrenomedullin were necessary to achieve its effects in comparison with plasma adrenomedullin concentrations. However, local adrenomedullin levels in vascular tissue may be much higher than plasma adrenomedullin concentrations, because adrenomedullin has recently been shown to be synthesized in and secreted from vascular endothelial cells²⁰ and smooth muscle cells.²¹ Furthermore, recently, plasma levels of adrenomedullin have been reported to be increased in patients with hypertension.²² Very recently, Jougasaki et al²³ reported an approximately fourfold increase in plasma levels of adrenomedullin in patients with congestive heart failure.

Inducible NO synthase activity is induced in blood vessel wall and cultured VSMCs by endotoxins and cytokines.⁵ Joly et al⁶ demonstrated that in vivo balloon injury induced NO synthase activity in rat carotid arteries, even in the absence of endothelium. Hansson et al²⁴ also reported that arterial smooth muscle cells in the neointima formed after deendothelializing balloon injury of the rat carotid artery expressed the cytokine-inducible isoform of NO synthase. On the other hand, reduced endothelial NO–mediated vasodilatation has been demonstrated in hypertensive animals and human subjects.²⁵ Thus, NO might be produced by vascular tissue under various pathological conditions and modulate contractility and cellular proliferation of vascular tissue.

Taken together with those observations, our results suggest that adrenomedullin increases NO production by cytokine-stimulated VSMCs in certain pathological states such as hypertension, atherosclerosis, and heart failure. However, further studies are required to clarify the role of adrenomedullin in NO synthesis in vascular tissue in vivo.

	Acknowledgments

 
This study was supported by the Ministry of Education, Science, Sports and Culture, Japan (No. 5670632).

Received November 1, 1995; first decision December 18, 1995; accepted February 13, 1996.

	References

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