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(Circulation. 1996;94:2560-2565.)
© 1996 American Heart Association, Inc.

Articles

Adrenomedullin Augments Inducible Nitric Oxide Synthase Expression in Cytokine-Stimulated Cardiac Myocytes

Uichi Ikeda, MD; Toshiko Kanbe; Yasuhiro Kawahara, MD; Mitsuhiro Yokoyama, MD; Kazuyuki Shimada, MD

the Department of Cardiology, Jichi Medical School, Tochigi (U.I., T.K., K.S.), and the First Department of Internal Medicine, Kobe University School of Medicine, Kobe (Y.K., M.Y.), Japan.

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

	Abstract

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Background Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes.

Methods and Results We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1ß (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1ß–stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10^-10 to 10^-6 mol/L). The adrenomedullin-induced nitrite production by interleukin-1ß–stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1ß–induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene–related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin.

Conclusions These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.

Key Words: adrenomedullin • interleukins • endothelium-derived factors • adenosine • myocardium

	Introduction

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Nitric oxide 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} Both constitutive and inducible pathways of NO synthesis probably have a biological role in the mammalian heart.^{3 4 5} Studies in rats have demonstrated that the myocardium expresses iNOS after treatment with cytokines. This suggests that induction of iNOS expression in the heart may cause the specific cardiac dysfunction observed in endotoxin shock and certain immunological and inflammatory conditions, including post–cardiac transplantation, myocarditis, and ischemia-reperfusion injury, in which various cytokines have been implicated.^{6 7}

Adrenomedullin, a potent endogenous vasodilating peptide, has recently been isolated from the acid extract of human pheochromocytoma.⁸ This peptide, consisting of 52 amino acids, has one intracellular disulfide bond and shows 20% homology with CGRP. Adrenomedullin injected intravenously has a potent and long-lasting hypotensive effect in rats.⁹ Recently, Ishizaka et al¹⁰ and Eguchi et al¹¹ demonstrated that adrenomedullin stimulates cAMP formation in rat vascular smooth muscle cells. Adrenomedullin thus acts directly on vascular smooth muscle and modulates vascular contractility and metabolism. However, there has been no report concerning the effects of adrenomedullin on cardiac myocytes, although the presence of adrenomedullin receptors has been shown in the heart,¹² and high plasma levels of adrenomedullin have been reported in patients with various cardiovascular disorders, including congestive heart failure.^{13 14} In this study, we investigated the effects of adrenomedullin on NO synthesis in cultured neonatal rat cardiac myocytes.

	Methods

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Materials
Recombinant rat adrenomedullin and human CGRP[8-37] were purchased from Peptide Institute Inc. Recombinant human interleukin-1ß was a gift from Otsuka Pharmacy. Anti–rat iNOS antibody was a gift from Dr H. Esumi (National Cancer Institute [Japan]). db-cAMP and IBMX were from Sigma Chemical Co. All other chemicals used were of the highest grade commercially available.

Culture of Cardiac Myocytes
Cardiac myocytes were prepared from ventricles of 1-day-old Sprague-Dawley rats as described previously.¹⁵ Briefly, after dissociation with 0.25% trypsin, cell suspensions were washed with DMEM supplemented with 10% FCS and centrifuged at 500g for 10 minutes. The centrifuged cells were then resuspended in 10% FCS containing DMEM. For selective enrichment of cardiac myocytes, the dissociated cells were preplated for 1 hour, during which time nonmyocytes readily attached to the bottom of the culture dish. The resulting suspension of myocytes was plated onto 24-well dishes at a density of 1x10⁶ cells/mL. Thymidine (0.6 mg/mL) was added during the first 72 hours to prevent proliferation of nonmyocytes. Using this method, we routinely obtained enriched cultures containing >95% myocytes, as assayed by immunofluorescence staining with an anti–myosin heavy chain antibody.¹⁶

These experiments were performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986, published by Her Majesty's Stationery Office, London.

Measurement of Nitrite
NO production by the cultured cells was determined by measuring the nitrite contents of the culture media. Cardiac myocytes plated in 24-well dishes were incubated in DMEM containing 0.5% FCS at 37°C. The nitrite contents of the culture media 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).¹⁷ 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 a washing, cells were dissolved in 0.2 mL 1% SDS and used for protein assay (BCA protein assay kit); BSA was used as a standard. Nitrite levels were corrected by protein levels, and data are shown as nmol/mg protein.

Assay for iNOS mRNA
Total RNA was extracted from cardiac myocytes plated in 100-mm culture dishes by the acid guanidinium thiocyanate–phenol-chloroform method, and 30-µg aliquots were subjected to electrophoresis on 1% agarose gels and transferred onto nylon filters. The filters were then hybridized with a random-primed ³²P-labeled mouse macrophage iNOS cDNA probe for 24 hours¹⁸ and washed twice with an aqueous solution of 150 mmol/L NaCl/15 mmol/L sodium citrate/0.1% SDS at 65°C. The filters were exposed to Kodak XAR-5 film for 1 to 2 days at -70°C with one intensifying screen; the results were quantified by densitometric scanning.

Assay for iNOS Protein
The expression of iNOS protein was analyzed by immunoblotting with an anti-iNOS antibody as described previously.¹⁹ Briefly, cardiac myocytes 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 PMSF, and 1 mol/L dithiothreitol and were sonicated. The homogenates were then centrifuged at 100 000g for 20 minutes, and the supernatants (60 µg protein) were subjected to 10% SDS-PAGE. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with anti-iNOS antibody 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.

Measurement of cAMP
For determination of intracellular cAMP levels, 0.5 mmol/L 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 in 24-well dishes with adrenomedullin for 60 minutes, cardiac myocytes were immediately immersed in 0.2 mL of 0.1N HCl to stop the reaction. Cells were then collected into glass tubes with a rubber policeman, boiled for 5 minutes, and then 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 they were assayed for cAMP content. The pellets were dissolved in 0.2 mL of 1% SDS and kept at 4°C until protein assay. Intracellular cAMP contents were measured with a commercial enzyme immunoassay kit with the manufacturer's high-sensitivity acetylation protocol (Amersham). The lower limit of detection was 2 fmol/well. The values were normalized to protein content of each well.

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

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Adrenomedullin on Nitrite Production
First, we investigated the effects of interleukin-1ß on nitrite production by cardiac myocytes. As reported previously,²⁰ addition of interleukin-1ß (10 ng/mL) to the cultures for 24 hours increased nitrite production by cardiac myocytes in a time-dependent manner. This interleukin-1ß–stimulated nitrite accumulation was significantly increased by simultaneous treatment of the cells with adrenomedullin. After a 24-hour incubation, the level of cytokine-stimulated nitrite accumulation in the presence of adrenomedullin (10^-7 mol/L) was about twice that in the absence of adrenomedullin.

Fig 1 shows the dose-response effect of adrenomedullin on nitrite production. Incubation with adrenomedullin for 24 hours increased interleukin-1ß–induced nitrite production by rat cardiac myocytes 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 (15K): [in this window] [in a new window]   Figure 1. Dose-dependent effects of adrenomedullin on nitrite accumulation. Cardiac myocytes were incubated for 24 hours with (solid columns) or without (open columns) 10 ng/mL interleukin-1ß in the presence of various concentrations of adrenomedullin (10-10 to 10-6 mol/L). Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. Data represent mean±SEM (n=4). *P<.05, **P<.01 compared with control cells without adrenomedullin.

Temporal Analysis of Stimulatory Action of Adrenomedullin
As shown in Fig 2, 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 2. Temporal analysis of the stimulatory action of adrenomedullin. Cardiac myocytes were stimulated for 24 hours with 10 ng/mL interleukin-1ß. Adrenomedullin (10-7 mol/L) was added either simultaneously with or 3, 6, or 12 hours after interleukin-1ß stimulation. Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. *P<.05, **P<.01 compared with control cells, indicated as (-).

Effects of Adrenomedullin on iNOS mRNA and Protein Expression
Since the temporal analysis described above strongly suggested that adrenomedullin increased interleukin-1ß–induced NO production at the level of iNOS expression, we examined whether adrenomedullin actually induced increases in iNOS mRNA levels in cardiac myocytes. As shown in Fig 3, unstimulated cells did not express iNOS mRNA. Incubation with interleukin-1ß for 24 hours resulted in induction of iNOS mRNA expression, and its expression was augmented in the presence of adrenomedullin.

View larger version (81K): [in this window] [in a new window]   Figure 3. Expression of iNOS mRNA in cardiac myocytes. Left, Northern blot depicting the iNOS mRNA expression. Cells were incubated for 24 hours with interleukin-1ß (10 ng/mL) and adrenomedullin (10-8, 10-7 mol/L). Total RNA was isolated and hybridized with 32P-labeled mouse macrophage iNOS (top) and 18S rRNA (bottom) cDNA probes. Lane 1, control; lane 2, interleukin-1ß; lane 3, interleukin-1ß plus 10-8 mol/L adrenomedullin; lane 4, interleukin-1ß plus 10-7 mol/L adrenomedullin. Right, Densitometry values (in arbitrary densitometry units) of iNOS mRNA in Northern blot (left). Values shown were corrected for unequal loading by use of the density of the corresponding 18S band. Data represent means of two separate experiments.

Furthermore, the expression of iNOS protein was analyzed by immunoblotting with the anti–iNOS antibody. No immunoreactive band of iNOS was detected in unstimulated cardiac myocytes (Fig 4). The iNOS protein band with a molecular mass of about 125 kD appeared clearly after exposure to interleukin-1ß for 24 hours, and its accumulation was further increased in the presence of adrenomedullin.

View larger version (42K): [in this window] [in a new window]   Figure 4. Effects of adrenomedullin on iNOS protein accumulation. Cultured rat cardiac myocytes were incubated for 24 hours with interleukin-1ß (10 ng/mL) and adrenomedullin (10-8, 10-7 mol/L). Cell extracts were subjected to SDS-PAGE followed by immunoblot analysis with the anti–rat iNOS antibody. Positions of the molecular mass markers are indicated on 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, interleukin-1ß; lane 3, interleukin-1ß plus 10-8 mol/L adrenomedullin; lane 4, interleukin-1ß plus 10-7 mol/L 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 NO production. Previously, we observed that cAMP upregulates iNOS expression in rat cardiac myocytes,²¹ and it recently has been shown that adrenomedullin stimulates cAMP formation in rat vascular smooth muscle cells^{10 11} ; therefore, we speculate the involvement of a cAMP-dependent pathway in the effect of adrenomedullin. As shown in Fig 5, in the absence of db-cAMP, adrenomedullin significantly increased interleukin-1ß–induced nitrite accumulation. On the other hand, in the presence of db-cAMP (1 mmol/L), the interleukin-1ß–induced nitrite accumulation was further increased, but the stimulatory effect of adrenomedullin was abolished.

View larger version (14K): [in this window] [in a new window]   Figure 5. Effects of db-cAMP on nitrite accumulation in cardiac myocytes. 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 columns) or absence (open columns) of 1 mmol/L db-cAMP. Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. Data represent mean±SEM (n=4). *P<.05.

We then actually measured intracellular cAMP levels in cardiac myocytes treated with adrenomedullin. As shown in Fig 6, incubation with adrenomedullin for 1 hour increased intracellular cAMP levels in a dose-dependent manner (10^-9 to 10^-6 mol/L).

View larger version (13K): [in this window] [in a new window]   Figure 6. Dose-dependent effects of adrenomedullin on intracellular cAMP levels in cardiac myocytes. Cells were incubated for 1 hour with 10-9 to 10-6 mol/L adrenomedullin in the presence of 0.5 mmol/L IBMX added 30 minutes before the addition of adrenomedullin. Intracellular cAMP levels were measured as described in "Methods," and the values were normalized to the protein content per well. Data represent mean±SEM (n=4). *P<.05 compared with control cells, indicated as (-).

It has been shown that the human CGRP receptor antagonist CGRP[8-37] inhibits cAMP generation by rat vascular smooth muscle cells induced by rat adrenomedullin.^{10 22} We thus tested the effects of CGRP[8-37] on cAMP and NO generation by rat cardiac myocytes and found that addition of CGRP[8-37] to the culture dose-dependently (10^-8 to 10^-5 mol/L) inhibited both cAMP (Fig 7) and nitrite (Fig 8) accumulation induced by adrenomedullin.

View larger version (15K): [in this window] [in a new window]   Figure 7. Dose-dependent effects of CGRP receptor antagonist on intracellular cAMP levels in rat cardiac myocytes induced by adrenomedullin. Cells were incubated for 1 hour with 10-7 mol/L adrenomedullin and 10-8 to 10-5 mol/L CGRP[8-37] in the presence of 0.5 mmol/L IBMX. Intracellular cAMP levels were measured as described in "Methods." Data represent percent inhibition of adrenomedullin-induced cAMP accumulation by CGRP[8-37]. Data are mean±SEM of four samples.

View larger version (15K): [in this window] [in a new window]   Figure 8. Dose-dependent effects of CGRP receptor antagonist on nitrite generation by rat cardiac myocytes induced by adrenomedullin. Cells were incubated for 24 hours with 10 ng/mL interleukin-1ß and 10-7 mol/L adrenomedullin in the presence of various concentrations of CGRP[8-37] (10-8 to 10-5 mol/L). Nitrite levels in the culture medium were measured as described in "Methods." Data represent percent inhibition of adrenomedullin-induced nitrite accumulation by CGRP[8-37]. Data are mean±SEM of four samples.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Adrenomedullin is a peptide recently isolated from pheochromocytoma that has vasorelaxant and long-lasting hypotensive activities.⁹ This peptide has been shown to be present not only in human adrenal medulla but also in heart, lung, kidney, and circulating blood.^{23 24} In this study, we investigated whether adrenomedullin modulates NO synthesis in cardiac myocytes. Although adrenomedullin by itself showed no effect on NO production, it augmented interleukin-1ß–induced NO production in a dose-dependent manner. The stimulatory effect of adrenomedullin was significantly reduced when added several hours after interleukin-1ß, suggesting that the augmentation of NO production by adrenomedullin is due to induction of iNOS. Indeed, adrenomedullin increased the accumulation of iNOS mRNA and protein in interleukin-1ß–stimulated cardiac myocytes.

We obtained four pieces of evidence for a causal link between cAMP production and augmentation of NO synthesis by adrenomedullin in cardiac myocytes. First, we have observed that cAMP increases interleukin-1ß–induced NO production by cardiac myocytes.²¹ Second, adrenomedullin caused an increase in the cellular levels of cAMP (Fig 6). Third, the stimulatory effect of adrenomedullin on NO production was abolished in the presence of db-cAMP (Fig 5). Fourth, CGRP[8-37] inhibited both cAMP and NO generation induced by adrenomedullin (Figs 7 and 8). These results suggest that adrenomedullin augments NO production by cardiac myocytes, 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 iNOS mRNA levels in interleukin-1ß–stimulated cardiac myocytes. Changes in the transcription and/or in mRNA stability may account for the observed changes in mRNA levels. Very recently, in rat cardiac myocytes, Oddis et al²⁵ revealed that cAMP enhances iNOS mRNA stability after cytokine exposure. However, nuclear run-on experiments will also be necessary to assess rates of transcription of the iNOS gene.

Saturation analysis revealed a dissociation constant for adrenomedullin of 0.41±0.14x10^-9 mol/L (mean±SD) in rat heart.²⁶ According to Sakata et al²⁷ and Ichiki et al,²⁴ mean plasma concentrations of adrenomedullin in rats and humans are 3.6±0.3x10^-9 and 3.3±0.4x10^-9 mol/L (mean±SD), respectively. Under the present experimental conditions, physiological concentrations of adrenomedullin were sufficient to achieve its effects in comparison with plasma adrenomedullin concentrations. In addition, local adrenomedullin levels in heart tissues may be much higher than plasma adrenomedullin concentrations because adrenomedullin has been shown to be synthesized in the heart.^{23 24} Recently, Jougasaki et al¹³ reported an approximately fourfold increase in plasma levels of adrenomedullin in patients with congestive heart failure.

Several previous studies have shown that interleukin-1ß stimulates NO synthesis in cardiac myocytes by inducing iNOS expression.^{5 20 28 29 30 31} Depressed myocardial contractility in both animals and humans is known to occur under conditions associated with the release of cytokines, including endotoxin shock or treatment with endotoxins in vitro.³² Similarly, during antitumor therapy with cytokines or after exposure to cytokines in vitro, marked depression of cardiac function has been observed.^{33 34 35 36} Previously, Hosenpud et al³⁷ reported that interleukin-1ß had a negative inotropic effect on canine hearts in vivo mediated through NO production. Roberts et al²⁸ and we³⁸ reported that interleukin-1ß showed a suppressive effect on beating rate of cultured rat cardiac myocytes mediated via NO production. Evans et al³⁹ also demonstrated that interleukin-1ß modulated myocardial contraction via dexamethasone-sensitive NO production in isolated ferret papillary muscle preparations. Enhanced NO generation by interleukin-1ß leads to sustained reduced myocardial contractility, presumably via activation of soluble guanylate cyclase to generate cGMP, which has been shown to suppress myocardial contractility by decreasing cytoplasmic Ca²⁺ concentrations.⁴⁰ Very recently, the lethal effects of cytokine-induced NO on cardiac myocytes have been reported.⁴¹

Recent studies have shown the upregulation of interleukin-1 expression in allografts in an experimental acute cardiac rejection model,^{6 42} increased plasma levels of interleukin-1 in patients with acute myocardial infarction,⁴³ high levels of iNOS expression in failing human cardiac myocytes,⁴⁴ and increased plasma levels of nitrite in patients with congestive heart failure.⁴⁵ In addition, high plasma levels of adrenomedullin have been reported in patients with various cardiovascular disorders, including congestive heart failure^{13 14} and acute myocardial infarction.⁴⁶ These observations are consistent with the hypothesis that adrenomedullin acts as an endogenous enhancer of NO production by cytokine-stimulated myocardium in certain immunological and inflammatory conditions, including post–cardiac transplantation, myocarditis, ischemia-reperfusion injury, and congestive heart failure.

In the present study, we revealed for the first time that the heart is a target organ of adrenomedullin and that adrenomedullin augments NO synthesis in the heart under cytokine-stimulated conditions. However, further studies are required to determine whether the effects of adrenomedullin on NO production described here contribute to the development of cardiac dysfunction in the above pathological states.

	Selected Abbreviations and Acronyms

 

CGRP = calcitonin gene–related peptide db-cAMP = N2,2'-O-dibutyryl cAMP IBMX = isobutylmethyl xanthine iNOS = inducible NO synthase

	Acknowledgments

 
This study was supported by the Ministry of Education, Culture, and Science of Japan (grants 5670632 and 8670821).

Received April 2, 1996; revision received June 12, 1996; accepted June 20, 1996.

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