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Endocrinology Vol. 144, No. 5 1712-1717
Copyright © 2003 by The Endocrine Society

Aldosterone Inhibits Inducible Nitric Oxide Synthase in Neonatal Rat Cardiomyocytes

Tae-Yon Chun, Laura J. Bloem and J. Howard Pratt

Department of Medicine (T.-Y.C., J.H.P.) and the Veterans Affairs Medical Center (J.H.P.), Indiana University Medical School, Indianapolis, Indiana 46202; and Lilly Research Laboratories (L.J.B.), Eli Lilly and Co., Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Tae-Yon Chun, Ph.D., Department of Medicine, 541 Clinical Drive, Room 458, Indianapolis, Indiana 46202. E-mail: tchun{at}iupui.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In studies of animals, increases in aldosterone are associated with myocardial necrosis and fibrosis, and treatment with spironolactone, an antagonist of aldosterone, improved clinical outcomes in patients with heart failure. In the present study, we explored nitric oxide (NO), a signaling molecule involved in cardiac function, as a potential mediator of aldosterone’s effects on the heart. Levels of both inducible NO synthase (iNOS) and NO from isolated rat neonatal cardiomyocytes pretreated with IL-1 were found to be decreased with exposure to aldosterone or dexamethasone in a dose-dependent manner. Spironolactone increased iNOS expression and prevented inhibition by aldosterone, consistent with a mineralocorticoid receptor-mediated mechanism for iNOS down-regulation. Aldosterone had no effect on iNOS mRNA levels, indicating a posttranscriptional mechanism for the inhibition of iNOS. Neutralization of TGF-ß1 using a specific antibody reversed aldosterone-dependent iNOS and NO down-regulation. In summary, aldosterone inhibited IL-1- induced iNOS expression posttranscriptionally by a TGF-ß1-dependent mechanism. The decrease in NO synthesis could have relevance to known cardiac effects of aldosterone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW WELL established that increases in aldosterone can adversely affect the integrity of the myocardium. The mineralocorticoid receptor (MR) where aldosterone binds to activate transcription has been identified in cardiomyocytes of several different species (1, 2, 3). In a hypertensive rat model (4) and a nitric oxide (NO)-deficient/angiotensin II-treated rat model (5), treatment with an MR antagonist prevented the development of myocardial necrosis. An increase in the level of aldosterone has also been shown to result in an accumulation of collagen in myocardium (6). In clinical studies, cardiac hypertrophy occurred in patients with primary aldosteronism more often than in patients with essential hypertension with a similar blood pressure (7). Finally, an important relationship of aldosterone to cardiac function was underscored by the results of a large clinical trial of congestive heart failure where small doses of the MR antagonist, spironolactone, substantially reduced cardiovascular-related morbidity and the risk of death (8), a response again unrelated to a reduction in blood pressure.

In the latter study, spironolactone may have blocked an effect of aldosterone to compromise cardiac function by decreasing myocardial compliance and contractility from a physical alteration of the myocardium, or from factors with more immediate influences on function. In the present study, we looked for an effect of aldosterone on the level of NO, a signaling molecule with a range of actions within the cardiovascular system. NO could potentially mediate any of the known effects of aldosterone excess on the heart. We found that aldosterone significantly decreased NO synthesis by inducible NO synthase (iNOS).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The neonatal rat cardiomyocyte was the cell type used in all the studies. The protocol for use of animals was approved by the Institutional Animal Care and Use Committee of Eli Lilly and Co. (Indianapolis, IN). The cells were prepared by stepwise enzymatic dissociation. One- to 3-d-old neonatal Sprague Dawley rats (Harlan, Indianapolis, IN) were decapitated and the cardiac ventricles excised. Each ventricle was cut into 15–20 pieces and washed with Hanks’ buffer (Life Technologies, Inc., Gaithersburg, MD) four times. Trypsin digestion buffer (1 mg/ml trypsin, 5 mM HEPES, 0.02 mg/ml deoxyribonuclease I) was added, and after 5–10 min of slow stirring, cell suspensions were transferred to a fresh tube with horse serum to inactivate trypsin. The digestion and collection step was repeated to maximize the cell harvest. Cells were harvested by spinning at 1000 rpm for 10 min and preplated for 2 h at a density of 12–15 million cells/100-mm culture dish to attach the nonmyocytes. The nonadherent cells were cultured in cardiomyocyte media (DMEM/F12 media with 2 g/liter BSA, 3 mM sodium pyruvate, 15 mM HEPES, 100 µM ascorbic acid, 1 µg/ml insulin, 1 µg/ml transferrin, 10 ng/ml sodium selenite, and 100 µg/ml ampicillin) with 5% horse serum and 0.1 mM of bromodeoxyuridine. After 24 h, the medium was changed to serum-free cardiomyocyte media. The enriched cardiomyocyte preparation was routinely checked for purity using routine microscopy; if there was less than 90% nonbeating cells, the preparation was not used. For iNOS induction, recombinant murine IL-1ß (IL-1; 2 ng/ml; R\|[amp ]\|D Systems, Minneapolis, MN) was added for 18 h. For neutralization of TGF-ß1 activity, antihuman TGF-ß1 antibody (5 µg/ml; R&D Systems) was added to the culture. The dosage was selected based on our prescreening assay and the recommendations from the manufacturer.

RNA isolation and reverse transcription
Total RNA was extracted from differentially treated rat cardiomyocytes by RNA isolation kit (Gentra, Minneapolis, MN). One microgram of total RNA was reverse transcribed in the mixture of 20 pM oligo (deoxythymidine)18, 0.5 mM deoxynucleotide triphosphates, 50 U of Moloney murine leukemia virus-reverse transcriptase (RT) (Life Technologies, Inc.) in 1x RT buffer (Life Technologies, Inc.) at 37 C for 2 h. The enzyme was inactivated by heating the reaction tube for 5 min at 95 C.

Semiquantitative PCR
PCR was performed with the mixture of 5 µl of RT reaction, 0.2 mM deoxynucleotide triphosphates, 0.4 µM of sense and antisense oligos, 2 U of Taq DNA polymerase (Sigma, St. Louis, MO). The amplifications were performed for 45 sec at 94 C, 45 sec at 60 C, and 90 sec at 72 C. Sense primer, 5'-ATGGAACAGTATAAGGCAAACACC-3' and antisense primer, 5'-GTTTCTGGTCGATGTCATGAGCAAAGG-3' yielded 220 bp of iNOS message. As a reaction control, actin message was amplified by sense primer 5'-CGTAAAGACCTCTATGCCAA-3' and antisense primer 5'-AGCCATGCCAAATGTCTCAT-3', which yielded 349 bp of actin.

iNOS Western analysis
The iNOS protein level was analyzed by Western analysis. Thirty to 40 µg of whole-cell protein samples were separated in 7.5% acrylamide gel by SDS-PAGE. After transferring to Immobilon-P transfer membrane (Millipore Corp., Bedford, MA), 10% milk in TBST buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 0.1% Tween 20) was added to block the nonspecific binding sites. Antihuman iNOS antibody (Transduction Laboratories, Inc., Lexington, KY) that cross-reacts with rat iNOS in 5% skim milk in TBST (1:2000 dilution) was added for 2 h at room temperature. After extensive washing with TBST, horseradish peroxidase linked antimouse antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) at 1:3000 dilution was added for 2 h. After washing, target protein bands were detected by the ECL detection system (Amersham Pharmacia Biotech).

Osteopontin Western analysis
The secreted form of osteopontin in cardiomyocyte culture media was analyzed after the media were concentrated 100-fold by Amicon 30 concentrator (Millipore Corp.). The concentrated media (20 µl) were run on 7.5% acrylamide gel as above. Antirat osteopontin antibody [MPIIIB10 (1), DSHB, Ames, IA] at 1:50 dilution in 5% milk in TBST was used to detect secreted form of osteopontin followed by secondary reactions and detection.

Nitrite quantification
Formation of NO in cultured rat cardiomyocytes was determined from the accumulation of nitrite (a stable breakdown product of NO) in the culture medium. The media, 18 h after cytokine treatment, were collected and centrifuged to remove cell debris. Cleared media were mixed with Griess reagent (Molecular Probes, Inc., Eugene, OR) and absorbance at 548 nm was measured spectrophotometrically. Nitrite concentration was measured using a standard curve of known concentration of NaNO2 provided in the kit.

Statistical analyses
A one-way ANOVA analysis using SAS software (SAS Institute, Cary, NC) was used to analyze the nitrite concentrations. The results are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Murine IL-1ß (IL-1) has been shown to cause induction of iNOS and NO synthesis in the rat cardiomyocyte (9). Thus, quiescent cardiomyocytes in serum-free media were exposed to IL-1 for 18 h in the presence or absence of steroids and spironolactone, and the nitrite level in the media was measured. Both aldosterone and dexamethasone, a glucocorticoid receptor (GR) agonist, significantly decreased nitrite levels in a dose-dependent manner (P = 0.003 and 0.0001 for aldosterone and dexamethasone, respectively, at 100 nM; Fig. 1AGo). Spironolactone, on the other hand, appeared to increase levels to above control values, although this did not reach significance. There was no detectable NO in the uninduced cells. iNOS protein levels in the presence of increasing concentrations of aldosterone, spironolactone, and dexamethasone are shown in Fig. 1BGo. Both aldosterone and dexamethasone decreased iNOS protein in a dose-dependent manner, whereas spironolactone had the reverse effect.



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  		Figure 1. The effects of aldosterone (Aldo), spironolactone (SL), and dexamethasone (Dex) on the levels of nitrite (A) and iNOS protein (B) in cardiomyocytes after induction by IL-1. A1–A100, Aldo (1–100 nM); S1–S100, SL (1–100 nM); D1–D100, Dex (1–100 nM). Aldosterone significantly decreased nitrite levels at concentrations of 10 and 100 nM as did dexamethasone at all concentrations (1, 10, and 100 nM). The addition of spironolactone resulted in a stepwise increase in nitrite levels that were not, however, significant. For the nitrite levels, values are expressed as the mean ± SE with P values depicted above the appropriate bars; n = 3 experiments for each treatment group. ns, Not significant.

 
To examine whether an MR antagonist would block the effect of either aldosterone or dexamethasone, we coincubated cells with increasing concentrations of spironolactone in the presence of 1 nM aldosterone (the physiologic circulating level of aldosterone) or dexamethasone (Fig. 2Go, A and B). A dose as low as 1 nM of spironolactone effectively antagonized the activity of aldosterone, increasing both NO (P = 0.051 when compared with aldosterone alone) and iNOS protein levels in a dose-dependent manner. On the other hand, spironolactone did not antagonize dexamethasone’s effects even at concentrations of spironolactone that were 100-fold greater. Thus, aldosterone appeared to down- regulate iNOS by a specific MR-mediated pathway, whereas dexamethasone presumably affected iNOS expression through a GR-mediated mechanism.



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  		Figure 2. Effects of aldosterone (Aldo), spironolactone (SL), and dexamethasone (Dex) on the levels of nitrite (A) and iNOS protein (B) in IL-1-treated cardiomyocytes. When comparisons were made with a physiologic level of aldosterone (1 nM; A1), the addition of spironolactone at concentrations of 1, 10, and 100 nM (A1/S1-A1/S100) resulted in significant increases in nitrite levels. Spironolactone had no effect to increase nitrite levels in dexamethasone-treated cells (D1 vs. D1/S1-D1/S100). For the nitrite levels, values are expressed as the mean ± SE with P values depicted above the appropriate bars; n = 3 experiments for each treatment group. ns, Not significant.

 
Glucocorticoids have been shown to suppress cytokine-induced iNOS activity in microvascular endothelial cells and cardiomyocytes by increasing the expression of osteopontin, a matrix phosphoprotein (10). Thus, we measured the secreted form of osteopontin from concentrated media after 18 h of differentially treated cardiomyocytes using Western blot analysis (Fig. 3Go). A 100-nM concentration of dexamethasone significantly increased the secreted form of osteopontin, whereas the same concentration of aldosterone (or spironolactone) were without effects—aldosterone did not mediate effects on iNOS and NO through a GR-dependent osteopontin pathway.



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  		Figure 3. Effects of adding aldosterone (A), dexamethasone (D), and spironolactone (S) on osteopontin secretion after 18 h of treatment of the rat cardiomyocytes (C, control). Osteopontin is known to down-regulate iNOS synthesis in cardiomyocytes (10 ). Dexamethasone increased osteopontin synthesis, whereas aldosterone and spironolactone had no effect on synthesis.

 
Rat iNOS gene expression is regulated by various known transcription factors including nuclear factor {kappa}B (NF-{kappa}B; Ref.11). Recently spironolactone was shown to decrease NF-{kappa}B activity in an angiotensin II-induced renal damage model (12). We sought to explore the role if any of NF-{kappa}B by employing an inhibitor, N{alpha}-p-tosyl-L-lysine-chloromethyl- ketone (TLCK; Ref.13). Although pretreatment with 50 µM TLCK reduced overall iNOS protein synthesis (Fig. 4Go), the ability of aldosterone and dexamethasone to decrease nitrite and iNOS protein levels (and spironolactone to increase levels) were maintained (P < 0.0001). The results are consistent with a posttranscriptional effect of aldosterone that was downstream of NF-{kappa}B activation.



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  		Figure 4. The effects of TLCK, an inhibitor of NF-{kappa}B activity, on nitrite (A) and iNOS protein (B) levels; results expressed as the mean ± SE. Cells were pretreated with 50 µM TLCK for 1 h before IL-1 induction in the presence or absence of 100 nM aldosterone (Aldo), spironolactone (SL), or dexamethasone (Dex). With simultaneous exposure to TLCK, aldosterone and dexamethasone significantly decreased and spironolactone significantly increased nitrite levels when compared with the control group. For the nitrite levels, values are expressed as the mean ± SE with P values depicted above the appropriate bars; n = 3 experiments for each treatment group.

 
To further test where the iNOS gene regulation occurred, we performed semiquantitative RT-PCR (25 cycles) to assess generation of iNOS mRNA with aldosterone or spironolactone added at time zero. mRNA levels were increased by 6 h, but there was no difference in levels between control, aldosterone, and spironolactone groups (using 15 cycles of PCR gave similar results; Fig. 5AGo). On the other hand, iNOS protein levels and nitrite levels (measured after 18 h) changed as expected (Fig. 5Go, B and C). Furthermore, we found that adding either steroids or spironolactone 6 h after IL-1 stimulation, when transcriptional rates would have reached maximal levels (9, 14, 15), resulted in the same changes in iNOS protein synthesis that we saw when aldosterone, dexamethasone, or spironolactone was added at time zero (Fig. 6Go; the change from control values were significant at P < 0.0001 for aldosterone and dexamethasone and P = 0.007 for spironolactone).



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  		Figure 5. Semiquantitative RT-PCR of iNOS expression (panel A), iNOS protein levels (panel B), and nitrite levels (panel C). Cardiomyocytes were stimulated with IL-1 with and without the addition of aldosterone or spironolactone (C-, control media without IL-1 stimulation). iNOS expression was unaffected by the addition of 100 nM aldosterone (A; Aldo) or spironolactone (S; SL) at the transcriptional level 6 and 18 h after IL-1 induction, whereas protein and NO levels changed as expected with the additions of either aldosterone or spironolactone after 18 h (the change in NO levels was marginally significant). The findings are consistent with a posttranscriptional effect of aldosterone and spironolactone on iNOS expression. Semiquantitative RT-PCR was repeated three times, and only representative data are shown. For the secreted nitrite levels, the values are expressed as the mean ± SE with P values depicted above the appropriate bars (C vs. A and C vs. S); n = 3 experiments for each treatment group. The difference between C- and C was significant at P < 0.0001.

 


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  		Figure 6. Nitrite and iNOS protein levels after the addition of 100 nM of steroids or spironolactone 6 h after induction by IL-1. With maximal transcription occurring at least by 6 h (Fig. 5Go); the delayed addition of the steroids/spironolactone resulted in the same response as when added at time 0, thus further implicating a posttranscriptional effect of the steroids/spironolactone [both aldosterone (Aldo) and dexamethasone (Dex) significantly lowered and spironolactone significantly increased levels of nitrite compared with cells treated with IL-1 only (C)]. For the nitrite levels, values are expressed as the mean ± SE with P values depicted above the appropriate bars; n = 3 experiments for each treatment group.

 
We considered that the posttranscriptional regulation of iNOS could be mediated by TGF-ß1 because it is synthesized and secreted by cardiomyocytes in response to stimuli that lead to cardiac remodeling (16). We coincubated IL-1-stimulated, aldosterone- or spironolactone-treated (100 nM for both) cardiomyocytes with either anti-TGF-ß1 antibodies or recombinant human TGF-ß1 (Fig. 7Go). The IC50 of the TGF-ß1 antibody was approximately 5 µg/ml, the concentration used in the experiments. Recombinant TGF-ß1 decreased NO levels in controls (C vs. CT, P < 0.0001) and in the spironolactone-treated group (S vs. ST, P < 0.0001) but was without additional effect when added to aldosterone (A vs. AT). On the other hand, the addition of TGF-ß1 antibody to the aldosterone-treated group completely prevented the inhibition that was seen with aldosterone alone (A vs. A{alpha}T, P < 0.0001), consistent with an important dependence of TGF-ß1. TGF-ß1 antibody had no effect on the control group or the spironolactone-treated group. The same antibody could not reverse the dexamethasone effect (data not shown), probably because dexamethasone inhibits iNOS at multiple sites such as by decreasing transcription and increasing osteopontin.



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  		Figure 7. The effects of neutralization with specific antibody to TGF-ß1 ({alpha}T) and of adding recombinant TGF-ß1 (T) on nitrite (panel A) and iNOS protein (panel B) levels after IL-1 stimulation of cardiomyocytes in the presence or the absence of 100 nM aldosterone (A) or spironolactone (S). The addition of antibody alone was without effect on nitrite levels (C vs. C{alpha}T), whereas recombinant TGF-ß1 significantly decreased levels (C vs. CT). Adding TGF-ß1 antibody reversed the inhibition of nitrite production by aldosterone (A vs. A{alpha}T). For the nitrite levels, values are expressed as the mean ± SE with P values depicted above the appropriate bars; n = 3 experiments for each treatment group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that aldosterone decreased NO production by iNOS in the neonatal rat heart, a response similar to what was described previously in vascular smooth muscle cells (17). Although the relevance of this action of aldosterone is unclear, NO plays a pivotal role in the maintenance of cardiac function (18). The change in NO observed could be connected to the well-recognized deleterious cardiac effects of aldosterone or the salutatory effects of treating with spironolactone. Our studies were, however, limited to acute effects of aldosterone and the applicability to previous observations made from chronic exposure (in vivo animal studies or clinical studies) is unknown. Although we have not studied as extensively noncardiomyocyte cells, preliminary results suggest that iNOS in cardiac fibroblasts may also be inhibited by aldosterone, which might further alter cardiac integrity and function.

Although glucocorticoids were shown previously to decrease iNOS expression (Refs.19, 20 and the present study), aldosterone’s effects on iNOS may be mediated by a different pathway from that used by glucocorticoids because spironolactone reversed the effect of a physiologic level of aldosterone but had no effect on the response to dexamethasone. Also, the expression of osteopontin, a known mediator of inhibition of iNOS (10), was not increased by aldosterone (in contrast, dexamethasone markedly increased osteopontin expression).

Aldosterone did not affect the level of iNOS mRNA. Also, adding aldosterone 6 h after the induction of iNOS and after the majority of transcription would have been expected to take place resulted in full inhibition of iNOS expression and NO synthesis, and thus it seemed to inhibit by a posttranscriptional mechanism. In previous studies, aldosterone was shown to produce a rapid down-regulation of protein kinase C activity in cardiomyocytes consistent with a nongenomic effect (21). Furthermore, spironolactone did not antagonize the effect of aldosterone on PKC activity (at higher concentrations spironolactone also decreased PKC activity). In our study, the aldosterone effect on NO and iNOS was antagonized by spironolactone consistent with a genomic effect (Fig. 2Go). Furthermore, when we incubated with aldosterone for only 30 min (6 h post induction), the aldosterone had no effect on inhibition of iNOS synthesis (data not shown), and more time was required, which again was in keeping with a genomic as opposed to nongenomic effect.

We considered the role of TGF-ß1 as a potential mediator of aldosterone’s effects because it is expressed in the myocardium, particularly so under conditions of cardiac strain when iNOS would also be expressed (22). Also, aldosterone had previously been shown to increase TGF-ß1 expression in kidney (23). We indeed did find that neutralization with TGF-ß1 antibody eliminated inhibition of iNOS by aldosterone and that the addition of recombinant TGF-ß1 partially replicated the effect of aldosterone. In our studies, aldosterone did not increase TGF-ß1 mRNA or level of protein (data not shown), and thus we speculate that aldosterone caused the transformation of latent TGF-ß1 to the active form (24). It might be noted that angiotensin II increases TGF-ß1 expression in the heart, contributing, it is thought, to cardiac hypertrophy (25); based on the present observations, it would seem tenable to consider that angiotensin II might also affect TGF-ß1 levels indirectly through increased synthesis of aldosterone.

The relevance of aldosterone’s ability to decrease NO production is unclear. iNOS expression would be expected to occur under conditions where there was a dysfunctional state of the heart as might occur with ischemia, cardiomyopathy, sepsis, or congestive heart failure (18, 26, 27, 28). There is a substantial body of evidence demonstrating that large increases in NO, as might occur with induction of iNOS, reduce the contractile response to adrenergic agonists (29, 30, 31). On the other hand, a requisite lower level of NO is important for maintaining normal cardiac function with either too little or too much NO impairing cardiac performance (18). Thus, the effect of aldosterone to reduce iNOS expression could under certain conditions lower NO to where it would result in a loss of optimal cardiac function, whereas spironolactone would restore function. Indeed, in an endothelial NO synthase knockout mouse model with ischemia/reperfusion injury, there was an increase in NO production and iNOS expression (32); the additionally generated NO from iNOS appeared to lead to an important adaptation. Finally, there is at least one human study where an aldosterone antagonist appeared to increase bioavailable NO (33). Spironolactone given to patients with congestive heart failure showed an improvement in NO-mediated arterial vasodilatation (as evidenced by a greater response to administered acetylcholine).

In conclusion, the current studies show that aldosterone inhibits iNOS in the neonatal rat cardiomyocyte. The response to aldosterone appears to require MR, occurs posttranscriptionally, and is dependent on TGF-ß1. Aldosterone-mediated inhibition of iNOS could aggravate cardiac function under conditions such as occur with heart failure.


   Acknowledgments
 
We thank Xushan Wang for his excellent technical assistance. The antiosteopontin antibody [MPIIIB10 (1)] developed by Drs. M. Solursh and A. Franzen was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA).


   Footnotes
 
Support was provided by NIH Grants R01-HL-35795 and R01-HL-67360, by a Merit Review grant from the U.S. Department of Veterans Affairs, and by an Indiana University School of Medicine Biomedical Research grant.

Abbreviations: GR, Glucocorticoid receptor; IL-1, murine IL-1ß; iNOS, inducible NO synthase; MR, mineralocorticoid receptor; NF-{kappa}B, nuclear factor {kappa}B; NO, nitric oxide; RT, reverse transcriptase; TLCK, N{alpha}-p-tosyl-L-lysine-chloromethyl-ketone.

Received September 11, 2002.

Accepted for publication January 8, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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