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(Circulation. 2005;111:51-57.)
© 2005 American Heart Association, Inc.

Heart Failure

Aldosteronism and a Proinflammatory Vascular Phenotype

Role of Mg²⁺, Ca²⁺, and H₂O₂ in Peripheral Blood Mononuclear Cells

Robert A. Ahokas, PhD; Yao Sun, MD, PhD; Syamal K. Bhattacharya, PhD; Ivan C. Gerling, PhD; Karl T. Weber, MD

From the Departments of Obstetrics and Gynecology (R.A.A.), Surgery (S.K.B.), and Divisions of Cardiovascular Diseases (Y.S., K.T.W.) and Endocrinology and Metabolism (I.C.G.), Department of Medicine, University of Tennessee Health Science Center, Memphis, Tenn.

Correspondence to Karl T. Weber, MD, Division of Cardiovascular Diseases, University of Tennessee Health Science Center, 920 Madison Ave, Suite 300, Memphis, TN 38163. E-mail KTWeber{at}utmem.edu

Received August 5, 2004; revision received September 10, 2004; accepted September 29, 2004.

	Abstract

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Background— Chronic, inappropriate (relative to dietary Na⁺) elevations in circulating aldosterone, such as occur in congestive heart failure, are accompanied by a proinflammatory vascular phenotype involving the coronary and systemic vasculature. An immunostimulatory state with activated peripheral blood mononuclear cells (PBMCs) precedes this phenotype and is induced by a fall in cytosolic free [Mg²⁺]_i and subsequent Ca²⁺ loading of these cells and transduced by oxidative/nitrosative stress.

Methods and Results— We sought to further validate this hypothesis in rats with aldosterone/1%NaCl treatment (ALDOST) by using several interventions as cotreatment: a Mg²⁺-supplemented diet; amlodipine, a CCB; and N-acetylcysteine, an antioxidant. Blood samples were obtained at weeks 1 to 4 of ALDOST to monitor [Mg²⁺]_i, [Ca²⁺]_I, and H₂O₂ production in PBMCs. Coronal ventricular sections were examined for invading inflammatory cells and 3-nitrotyrosine labeling, a marker of oxidative/nitrosative stress. In response to ALDOST and compared with untreated controls, we found an early and persistent reduction in [Mg²⁺]_i with a subsequent rise in [Ca²⁺]_i and H₂O₂ production, each of which was either attenuated or abrogated by the Mg²⁺-supplemented diet and by N-acetylcysteine, whereas amlodipine prevented Ca²⁺ loading and an altered redox state. Cotreatment with these interventions either markedly attenuated or prevented the appearance of the proinflammatory coronary vascular phenotype and the presence of 3-nitrotyrosine in invading inflammatory cells.

Conclusions— We suggest that the immunostimulatory state that appears during aldosteronism and leads to a proinflammatory coronary vascular phenotype is induced by a fall in [Mg²⁺]_i with Ca²⁺ loading of PBMCs and is transduced by H₂O₂ production in these cells.

Key Words: calcium • stress • pathology • aldosterone • magnesium

	Introduction

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In both humans and rats, inappropriate (relative to dietary Na⁺) elevations in circulating aldosterone are accompanied by a proinflammatory/fibrogenic vascular phenotype that involves intramural arteries of the heart and systemic organs.^1–5 In a rat model of aldosterone/salt treatment (ALDOST), the appearance of such vascular remodeling is Na⁺- and time-dependent and is prevented by cotreatment with spironolactone, an aldosterone receptor antagonist, in either nondepressor or depressor dosage.⁶ Lesions include ED1-positive monocytes/macrophages and CD4-positive lymphocytes that first invade the perivascular space of involved vessels at week 4 of ALDOST.^4,7,8

We have previously found evidence of oxidative/nitrosative stress in these inflammatory cells, including an activation of gp91^phox, an NADPH oxidase subunit, and the presence of 3-nitrotyrosine, a product of the reaction between nitric oxide and superoxide; activation of a redox-sensitive nuclear transcription factor-B; and upregulated mRNA expression of a proinflammatory mediator cascade regulated by nuclear transcription factor-B and which includes intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and tumor necrosis factor-.⁷ Cotreatment with an antioxidant⁷ or with agents that negatively regulate the transcription of inflammatory response genes⁹ is vasculoprotective in the setting of chronic mineralocorticoid excess.

Peripheral blood mononuclear cells (PBMCs) are activated weeks before the appearance of these vascular lesions. Included here is an increased production of H₂O₂ by monocytes and lymphocytes and an upregulated, differential expression of genes for antioxidant defenses, CC and CXC chemokines and their receptors, such cytokines as interleukin-1ß, and lymphocyte activation.^10,11 This immunostimulatory state appears in the absence of prior organ injury and therefore without apparent exposure to self-antigen. Its pathogenesis has been attributed to H₂O₂, which is known to act as an intracellular messenger to mimic antigen–antigen receptor binding.¹² Mechanisms responsible for the induction of oxidative/nitrosative stress in PBMCs during ALDOST appear to be linked to iterations in their divalent cation composition, specifically [Mg²⁺]_i and [Ca²⁺]_i.^10,11

Our current working hypothesis suggests PBMC activation in ALDOST is induced by a reduction in biologically active, cytosolic free [Mg²⁺]_i and subsequent Ca²⁺ loading of these cells and that it is transduced by oxidative/nitrosative stress. Further evidence in support of this contention, albeit circumstantial, is the response to cotreatment with spironolactone, which attenuates Ca²⁺ loading and the induction of oxidative/nitrosative stress in these cells and their invasion of the coronary vasculature.^7,11 The present study was undertaken in PBMCs harvested from rats receiving 1 to 4 weeks ALDOST to further address the role of iterations in [Mg²⁺]_i and [Ca²⁺]_i in altering their redox state. The following interventions were examined as cotreatment with ALDOST: a Mg²⁺-supplemented diet to prevent the fall in PBMC [Mg²⁺]_i and rise in [Ca²⁺]_i^13,14; an L-type Ca²⁺ channel blocker (CCB; amlodipine) to attenuate the rise in intracellular Ca²⁺ in these immune cells and which has been shown to ameliorate the appearance of proinflammatory coronary lesions in the setting of chronic mineralocorticoid excess^15,16; and an antioxidant (N-acetylcysteine [NAC]) to attenuate oxidative/nitrosative stress in these inflammatory cells.⁷ Blood samples were obtained at weeks 1 to 4 of ALDOST to monitor [Mg²⁺]_i and [Ca²⁺]_i and to determine lymphocyte H₂O₂ production in PBMCs. The intramural coronary artery vasculature found in coronal sections of right and left ventricular tissue was examined for evidence of invading inflammatory cells and oxidative/nitrosative stress as reflected by 3-nitrotyrosine labeling.

	Methods

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Animals
Eight-week-old male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, Ind) were used in this study. Five groups were studied: (1) untreated, unoperated age-/gender-matched rats, which served as controls; (2) uninephrectomized rats given standard chow (20 to 40 mg/kg Mg²⁺) together with 1% NaCl/0.4% KCl in drinking water and aldosterone (0.75 µg/h) administered by implanted minipump (ALDOST); (3) ALDOST plus a Mg²⁺-supplemented diet (Mg²⁺ content 40 to 60 mg/kg); (4) ALDOST plus cotreatment with an L-type CCB, (amlodipine, 10 mg · kg^–1 · d^–1 by gavage); and (5) ALDOST plus NAC (200 mg/kg QD IP). Each group was studied at 1 to 4 weeks of treatment. Animals were monitored for clinical appearance and body weight. Under anesthesia, blood (7 mL) was collected by cardiac puncture. PBMCs were isolated as previously reported.^10,11 In brief, cells were isolated by Histopaque 1077 centrifugation for 30 minutes at 400g. Cells were carefully aspirated, washed twice, and suspended in PBS. Hearts were removed, rinsed in cold saline solution, and frozen in isopentane with dry ice for immunohistochemistry. The study was approved by this institution’s Animal Care and Use Committee.

PBMC Cytosolic Free [Mg²⁺]_i and [Ca²⁺]_i
Cytosolic free [Mg²⁺]_i and [Ca²⁺]_i were measured as reported previously¹¹ with a modification of the ratiometric method and the fluorescent molecular probes mag-fura-2 and fura-2 (Molecular Probes), respectively. Briefly, PBMCs were washed and suspended in RPMI 1640 media containing 0.1% bovine serum albumin (pH 7.4). For loading the probes, the cells were incubated with mag-fura-2/AM (10 µmol/L) for Mg²⁺ or fura-2/AM (5 µmol/L) for Ca²⁺ for 1 hour at 37°C. After being washed 3 times with fresh RPMI 1640 media, the cells were suspended in analysis buffer consisting of (in mmol/L) NaCl 140, KCl 5, CaCl₂ 1.8, MgSO₄ 0.8, HEPES 15, and D-glucose 5 (pH 7.4) for the measurement of fluorescence with a Perkin-Elmer LS-50B spectrofluorometer. Fluorescence emission at 510 nm (slit width 10 nm) was recorded with excitation at 335 and 370 nm (slit width 10 nm) for the measurement of [Mg²⁺]_i in the cells loaded with mag-fura-2 after addition of 5 mmol/L EDTA and 5 mmol/L EGTA (R), after addition of 0.1% Triton X-100 (R_min), and after addition of 100 mmol/L MgSO₄ (R_max). [Mg²⁺]_i was calculated as follows: [Mg²⁺]_i (mmol/L)=K_dx[(R–R_min)S_f]/[(R_max–R)S_b], where K_d, the dissociation constant for Mg²⁺, is 1.5 mmol/L, and S_f and S_b are the fluorescence intensities at 370 nm with 0 Mg²⁺ and excess Mg²⁺, respectively. For measurement of [Ca²⁺]_i in the fura-2-loaded cells, fluorescence emission at 510 nm (slit width 7 nm) was recorded with excitation at 340 and 380 nm (slit width 10 nm), respectively, after addition of 10 mmol/L EGTA (R), after addition of 0.1% Triton X-100 (R_min), and after addition of 10 mmol/L CaCl₂ (R_max). [Ca²⁺]_i (nmol/L) was calculated with the same equation as for [Mg²⁺]_i using the Ca²⁺ dissociation constant (K_d) of 225 nmol/L, and S_f and S_b were the fluorescence intensities at 380 nm with 0 Ca²⁺ and excess Ca²⁺, respectively.

PBMC Hydrogen Peroxide Production
Lymphocyte H₂O₂ production was measured as reported previously.¹¹ Briefly, 0.1 mL of whole blood was incubated with 2,7-dichlorofluorescein diacetate (25 µmol/L, Molecular Probes) for 45 minutes at 37°C. After red blood cells were lysed with FACS lysing solution (Becton Dickinson), leukocytes were suspended in PBS (pH 7.4) for single-cell flow cytometric analysis with a FACS Caliber flow cytometer (Becton Dickinson). Lymphocytes were discerned by the combination of low-angle forward-scattered and right-angle scattered laser light. Fluorescent emission at 510 to 550 nm was recorded with excitation at 488 nm.

Immunohistochemistry (3-Nitrotyrosine)
Coronal cryostat sections (6 µm) were prepared, air-dried, fixed in 10% buffered formalin for 5 minutes, and washed in PBS for 10 minutes. Sections were then incubated with primary antibody against 3-nitrotyroxine at a dilution of 1:100 (Upstate Biotech) in PBS containing 1% BSA for 60 minutes. Sections were then washed in PBS for 10 minutes and incubated with IgG-peroxidase-conjugated secondary antibody (Sigma) with a dilution of 1:150, washed in PBS for 10 minutes, incubated with 0.5 mg/mL diaminobenzidine tetrahydrochloride 2-hydrate plus 0.05% H₂O₂ for 10 minutes, and again washed in PBS. Negative control sections were incubated with secondary antibody alone, stained with hematoxylin, dehydrated, mounted, and examined by light microscopy.⁷

Statistical Analysis
Values presented are mean±SEM. Data were statistically analyzed by 1-way ANOVA. Significant differences between individual means were determined with the post hoc Bonferroni multiple comparison test, where significance was assigned to P<0.05.

	Results

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Clinical Observations
At weeks 1 and 2 ALDOST, animals were active, eating, and gaining weight at levels comparable to controls. Anorexia, lethargy, and failure to gain weight were seen at weeks 3 and 4. Rats in the 3 intervention groups receiving ALDOST plus either Mg²⁺-supplemented diet, CCB, or NAC did not appear as ill as those receiving ALDOST alone. This is reflected in their body weights, which were less than controls at weeks 3 and 4 but greater than in those with ALDOST alone (Table).

View this table: [in this window] [in a new window]   Body Weights (g) During ALDOST and With Various Interventions

[Mg²⁺]_i and [Ca²⁺]_i Concentrations in PBMCs
In unoperated, untreated controls receiving standard chow with 20 to 40 mmol/kg Mg²⁺, the mean concentration of [Mg²⁺]_i in PBMCs was 349±17 µmol/L (Figure 1). This cytosolic free component of intracellular Mg²⁺ was significantly reduced throughout weeks 1 to 4 ALDOST. This fall in [Mg²⁺]_i was either attenuated by the Mg²⁺-supplemented diet or NAC but was not affected by CCB.

View larger version (14K): [in this window] [in a new window]   Figure 1. Cytosolic free Mg2+ concentrations ([Mg2+]i) in PBMCs harvested at weeks 1 to 4 of ALDOST are presented as open columns and compared with values for controls (dashed line). Each panel represents separate intervention as solid column: Mg2+-supplemented (Suppl) diet; amlodipine (CCB); and NAC. Mean±SEM (n=5 at all time points). *P<0.05 vs controls. P<0.05 vs ALDOST. See text for details.

PBMC [Ca²⁺]_i began to rise during week 2 and was statistically greater than in controls at weeks 3 and 4 ALDOST (Figure 2). This gradual rise in PBMC [Ca²⁺]_i was prevented with the Mg²⁺-supplemented diet, amlodipine, or NAC.

View larger version (14K): [in this window] [in a new window]   Figure 2. Cytosolic free Ca2+ concentration ([Ca2+]i) in PBMCs obtained at weeks 1 to 4 ALDOST are presented as open columns and compared with values for controls (dashed line). Each panel represents separate intervention as solid column: Mg2+-supplemented (Suppl) diet; amlodipine (CCB); and NAC. Mean±SEM (n=5 at all time points). *P<0.05 vs controls. P<0.05 vs ALDOST. See text for details.

Lymphocyte Hydrogen Peroxide Production
H₂O₂ production rose progressively in PBMCs harvested from rats during weeks 1 to 4 ALDOST and was significantly greater than controls at weeks 2 to 4 (Figure 3). In response to the Mg²⁺-supplemented diet, lymphocyte H₂O₂ production was increased at week 1 ALDOST but did not differ from controls at weeks 2 to 4. During cotreatment with amlodipine or NAC, lymphocyte production of H₂O₂ was attenuated compared with ALDOST and was not different from controls.

View larger version (15K): [in this window] [in a new window]   Figure 3. Hydrogen peroxide (H2O2) production by PBMCs, expressed as mean channel brightness (MCB), during weeks 1 to 4 of ALDOST are presented as open columns and compared with values for controls (dashed line). Each panel represents separate intervention as solid column: Mg2+-supplemented (Suppl) diet; amlodipine (CCB); and NAC. Mean±SEM (n=5 at all time points). *P<0.05 vs controls. P<0.05 vs ALDOST. See text for details.

Immunohistochemistry
Superoxide anions react with nitric oxide to form short-lived peroxynitrite and ultimately stable nitrotyrosine protein moieties. The expression of 3-nitrotyrosine, as detected by immunohistochemistry, was used as an in situ marker of oxidative/nitrosative stress in the heart (Figure 4A). In controls, 3-nitrotyrosine is expressed by coronary vascular endothelial and smooth muscle cells; it is not found in the perivascular space or in cardiomyocytes (panel A). In ALDOST rats, perivascular swelling and infiltration by inflammatory cells is seen surrounding intramural coronary arteries together with 3-nitrotyrosine labeling (Figure 4B). The Mg²⁺-supplemented diet or CCB attenuated the expansion of the perivascular space and cellular infiltration and the appearance of 3-nitrotyrosine staining (Figure 4C and 4D). NAC treatment completely prevented such vascular remodeling and appearance of 3-nitrotyrosine with ALDOST (Figure 4E).

View larger version (205K): [in this window] [in a new window]   Figure 4. Immunohistochemical labeling for 3-1nitrotyrosine. In normal rat heart, 3-nitrotyrosine is found solely in vascular endothelial and smooth muscle cells (A). At 4 weeks of ALDOST, 3-nitrotyrosine labeling is present in inflammatory cells found in perivascular space of intramural coronary arteries (B). Cotreatment with Mg2+-supplemented diet or amlodipine attenuated appearance of invading inflammatory cell response and accompanying 3-nitrotyrosine staining (C and D, respectively). Cotreatment with NAC prevented appearance of invading cells and 3-nitrotyrosine labeling in perivascular space (E). F, Negative control. Original magnification, x480.

	Discussion

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The present study yielded several major findings. First, the early and persistent reduction in PBMC [Mg²⁺]_i that accompanies ALDOST was attenuated by a Mg²⁺-supplemented diet and NAC but not by CCB. As we and others have previously reported,^10,11,13,17 chronic elevations in plasma aldosterone or deoxycorticosterone (DOC), another mineralocorticoid, inappropriate relative to dietary Na⁺, are associated with reduced lymphocyte [Mg²⁺]_i. Herein, we confirmed this response and documented its presence throughout 4 weeks of ALDOST. Mechanisms responsible for the early and sustained fall in [Mg²⁺]_i may be several fold. Hypermagnesuria accompanies aldosteronism and likely contributes to the fall in [Mg²⁺]_i.^18,19 Additionally, lymphocyte [Mg²⁺]_i is reduced in patients with primary aldosteronism, and in ex vivo studies of human lymphocyte, [Mg²⁺]_i fell in response to incubation with physiologically relevant concentrations of aldosterone.¹⁷ Delva et al¹⁷ suggested this Na⁺-dependent, aldosterone receptor–mediated response involves the transcription and synthesis of a putative Na⁺/Mg²⁺ exchanger; however, Mg²⁺ efflux was not measured in that study. In preventing the decline in [Mg²⁺]_i with NAC, findings of the present study suggest reactive oxygen species contribute to this response. Further studies are needed to elucidate the mechanisms responsible for the decline in [Mg²⁺]_i during ALDOST in rats are planned.

The fall in PBMC [Mg²⁺]_i that appears with ALDOST is accompanied by a subsequent rise in cytosolic free [Ca²⁺]_i in these cells. Increased monocyte and lymphocyte [Ca²⁺]_i controls numerous cell functions, including adhesion, motility, gene expression, and cell proliferation. Ca²⁺ loading of lymphocytes, as well as platelets and vascular smooth muscle cells, occurs in response to chronic DOC/salt treatment in rats^14,20,21 and with mineralocorticoid administration in humans.²² The fall in PBMC [Mg²⁺]_i seen with ALDOST could contribute to Ca²⁺ loading of these cells, because intracellular Mg²⁺ is a potent antagonist to Ca²⁺ entry.²³ The importance of reduced [Mg²⁺]_i in initiation of increased [Ca²⁺]_i and the induction of oxidative/nitrosative stress in PBMCs, together with an exaggerated immune cell response, has also been observed in rats fed a Mg²⁺-deficient diet.^24–28 PBMC activation, which appears during week 1 of such a diet, precedes inflammatory cell invasion of cardiac tissue by several weeks.²⁵ Herein, we were able to prevent the fall in [Mg²⁺]_i that appears with ALDOST by a Mg²⁺-supplemented diet. A reduced activity of Mg²⁺-dependent Na⁺/K⁺-ATPase may be contributory to the observed rise in PBMC [Ca²⁺]_i. DOC/salt treatment augments the number of Ca²⁺ channels and raises [Ca²⁺]_i in various cell populations.²⁹ An aldosterone-induced expression and increased activity of T-type Ca²⁺ channels has been reported in adrenal cells, cardiomyocytes, and prostate epithelial cells and is blocked by spironolactone.^30–32 Whether this occurs in PBMCs during ALDOST is unknown, but if so, it would enhance the transfer of extracellular Ca²⁺ directly to mitochondria via the endoplasmic reticulum and would contribute to the induction of oxidative/nitrosative stress in these cells. Mibefradil, a dual T- and L-type CCB, inhibits the proliferation of stimulated PBMCs,³³ reduces leukocyte adhesion by interfering with integrin signaling,³⁴ and is organ protective in rat models of chronic mineralocorticoid/salt excess.^35,36 In the present study, we found amlodipine, an L-type channel blocker, attenuates PBMC Ca²⁺ loading and the appearance of coronary vascular lesions. Other dihydropyridine CCBs have proved to be organ protective in rats with chronic mineralocorticoid excess.^15,16,37 Lymphocytes are otherwise nonexcitable cells and are where the membrane potential of T cells is primarily determined by both voltage- and Ca²⁺-dependent K⁺ channels, whose behavior resembles voltage-dependent Ca²⁺ channels.³⁸ Dihydropyridine CCBs inhibit Ca²⁺ entry in lymphocytes via their influence on Ca²⁺-activated K⁺ channels.³⁸ Nonetheless, enhanced PBMC Ca²⁺ entry occurs during ALDOST. We cannot discount a release of this cation from intracellular organelles. It has been reported that once present, oxygen and nitrogen metabolites can augment Ca²⁺ entry via voltage-dependent channels,³⁹ which is supported by our findings with NAC that abrogated PBMC Ca²⁺ loading during ALDOST.

Our second major finding is the induction of oxidative/nitrosative stress in PBMCs that accompanies increased [Ca²⁺]_i and which was evidenced by increased H₂O₂ production in circulating lymphocytes. Additional evidence of oxidative/nitrosative stress is the immunohistochemical detection of 3-nitrotyrosine we found in monocytes and lymphocytes invading the perivascular space of intramural coronary arteries. Reactive oxygen species, such as H₂O₂, serve as intracellular messengers in activating transcription of inflammatory cells, thus promoting their proinflammatory behavior.^12,40 We have previously reported on the early activation of PBMC transcriptome during ALDOST. This includes a differential expression of genes that encode an oxidative stress–inducible tyrosine phosphatase and such antioxidant defenses as superoxide dismutase and that are coupled to upregulated expression of proinflammatory genes and those in keeping with lymphocyte activation and autoreactivity.^10,11 The rise in lymphocyte H₂O₂ production during ALDOST reflects an imbalance between the generation of reactive oxygen and nitrogen species and their inadequate neutralization by likely depleted antioxidant defenses.

The fall in [Mg²⁺]_i and accompanying responses in intracellular Ca²⁺ and redox state observed in PBMCs during ALDOST were prevented or attenuated by dietary Mg²⁺ supplementation. Similar responses to Mg²⁺ supplementation have been reported in rodent and human platelets during chronic mineralocorticoid excess (inappropriate for dietary Na⁺ intake) and in rodent neutrophils in response to dietary Mg²⁺ deficiency.^14,22,28 The proinflammatory phenotype seen with dietary Mg²⁺ deficiency is prevented when such a diet is simultaneously made Ca²⁺-deficient.²³ A Mg²⁺-supplemented diet has been reported to attenuate endothelin-1 production by heart, aorta, and kidneys in rats receiving DOC/salt treatment.^41,42 This calcitropic hormone produced by endothelial cells is also released by parathyroid glands in response to reduced serum ionized Ca²⁺.⁴³ Herein, we found cotreatment with a CCB to attenuate PBMC Ca²⁺ loading during ALDOST. In blocking Ca²⁺ entry, amlodipine attenuated H₂O₂ production by these cells. Further evidence supporting the importance of Ca²⁺ loading in leading to an altered PBMC redox state is derived from our previous findings with spironolactone cotreatment, which prevented the rise in [Ca²⁺]_i and induction of oxidative stress, including H₂O₂ production, and attenuated the upregulation of transcriptome of the proinflammatory PBMC phenotype.¹¹ Our in vivo findings underscore the importance of an equilibrium between intracellular Mg²⁺ and Ca²⁺ in the generation of reactive oxygen and nitrogen species, which in physiologically relevant concentrations may represent a final common pathway in the activation of lymphocytes and monocytes. Left unbridled, marked increments in reactive oxygen and nitrogen intermediates would adversely affect cell survival.

Finally, interventions used in the present study either attenuated or abrogated the proinflammatory coronary vascular phenotype and the presence of 3-nitrotyrosine in invading inflammatory cells at 4 weeks of ALDOST. Sun et al⁷ previously reported that cotreatment with either an antioxidant, pyrrolidine dithiocarbamate, or spironolactone abrogates the appearance of oxidative/nitrosative stress and the number of inflammatory cells that invade the coronary vasculature at week 4 of ALDOST. Several clinical trials support the efficacy of aldosterone receptor antagonism, together with treatment with an ACE inhibitor and diuretic, in the overall management of symptomatic heart failure,^44,45 in which aldosteronism is accompanied by evidence of oxidative/nitrosative stress in plasma, PBMCs, and such diverse tissues as skeletal muscle and heart.^46–50 An integral feature of this systemic disorder is a catabolic state with simultaneous loss of lean tissue, fat, and bone and which eventuates in a wasting syndrome termed cardiac cachexia.⁵¹ We believe inappropriate chronic elevations in plasma aldosterone (relative to dietary Na⁺) contribute to this illness through an immunostimulatory state mediated by iterations in PBMC divalent cations and oxidative/nitrosative stress, a contention supported by findings of the present study.

	Acknowledgments

 
This work was supported in part by R01-HL67888 (Dr Sun), R01-DK62403 (Dr Gerling), R24-RR15373 (Dr Gerling), R21-DK55263 (Dr Gerling), and R01-HL73043 (Dr Weber) from the National Institutes of Health.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricle in experimental hypertension. Circ Res. 1990; 67: 1355–1364.[Abstract/Free Full Text]
   
2. Campbell SE, Diaz-Arias AA, Weber KT. Fibrosis of the human heart and systemic organs in adrenal adenoma. Blood Press. 1992; 1: 149–156.[Medline] [Order article via Infotrieve]
   
3. Robert V, Van Thiem N, Cheav SL, Mouas C, Swynghedauw B, Delcayre C. Increased cardiac types I and III collagen mRNAs in aldosterone-salt hypertension. Hypertension. 1994; 24: 30–36.[Abstract/Free Full Text]
   
4. Campbell SE, Janicki JS, Weber KT. Temporal differences in fibroblast proliferation and phenotype expression in response to chronic administration of angiotensin II or aldosterone. J Mol Cell Cardiol. 1995; 27: 1545–1560.[CrossRef][Medline] [Order article via Infotrieve]
   
5. Young M, Head G, Funder J. Determinants of cardiac fibrosis in experimental hypermineralocorticoid states. Am J Physiol. 1995; 269: E657–E662.[Medline] [Order article via Infotrieve]
   
6. Weber KT. From inflammation to fibrosis: a stiff stretch of highway. Hypertension. 2004; 43: 716–719.[Free Full Text]
   
7. Sun Y, Zhang J, Lu L, Chen SS, Quinn MT, Weber KT. Aldosterone-induced inflammation in the rat heart: role of oxidative stress. Am J Pathol. 2002; 161: 1773–1781.[Abstract/Free Full Text]
   
8. Rocha R, Rudolph AE, Frierdich GE, Nachowiak DA, Kekec BK, Blomme EA, McMahon EG, Delyani JA. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol. 2002; 283: H1802–H1810.
   
9. Iglarz M, Touyz RM, Viel EC, Paradis P, Amiri F, Diep QN, Schiffrin EL. Peroxisome proliferator-activated receptor- and receptor- activators prevent cardiac fibrosis in mineralocorticoid-dependent hypertension. Hypertension. 2003; 42: 737–743.[Abstract/Free Full Text]
   
10. Gerling IC, Sun Y, Ahokas RA, Wodi LA, Bhattacharya SK, Warrington KJ, Postlethwaite AE, Weber KT. Aldosteronism: an immunostimulatory state precedes the proinflammatory/fibrogenic cardiac phenotype. Am J Physiol Heart Circ Physiol. 2003; 285: H813–H821.[Abstract/Free Full Text]
    
11. Ahokas RA, Warrington KJ, Gerling IC, Sun Y, Wodi LA, Herring PA, Lu L, Bhattacharya SK, Postlethwaite AE, Weber KT. Aldosteronism and peripheral blood mononuclear cell activation: a neuroendocrine-immune interface. Circ Res. 2003; 93: e124–e135.[Abstract/Free Full Text]
    
12. Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat Immunol. 2002; 3: 1129–1134.[CrossRef][Medline] [Order article via Infotrieve]
    
13. Suzuki H, Sano H, Fukuzaki H. Decreased cytosolic free calcium concentration in lymphocytes of magnesium-supplemented DOCA-salt hypertensive rats. Clin Exp Hypertens A. 1989; 11: 487–500.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Kh R, Khullar M, Kashyap M, Pandhi P, Uppal R. Effect of oral magnesium supplementation on blood pressure, platelet aggregation and calcium handling in deoxycorticosterone acetate induced hypertension in rats. J Hypertens. 2000; 18: 919–926.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Aguas AP, Nickerson PA. Effect of verapamil on blood pressure and lesions in heart and kidney of rats made hypertensive by deoxycorticosterone (DOC). Am J Pathol. 1983; 110: 48–54.[Abstract]
    
16. Nickerson PA. A low dose of a calcium antagonist (nitrendipine) ameliorates cardiac and renal lesions induced by DOC in the rat. Exp Mol Pathol. 1984; 41: 309–320.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Delva P, Pastori C, Degan M, Montesi G, Brazzarola P, Lechi A. Intralymphocyte free magnesium in patients with primary aldosteronism: aldosterone and lymphocyte magnesium homeostasis. Hypertension. 2000; 35: 113–117.[Abstract/Free Full Text]
    
18. Horton R, Biglieri EG. Effect of aldosterone on the metabolism of magnesium. J Clin Endocrinol Metab. 1962; 22: 1187–1192.[Medline] [Order article via Infotrieve]
    
19. Chhokar VS, Sun Y, Bhattacharya SK, Ahokas RA, Myers LK, Xing Z, Smith RA, Gerling IC, Weber KT. Loss of bone minerals and strength in rats with aldosteronism. Am J Physiol Heart Circ Physiol. 2004; 287: H2023–H2026.[Abstract/Free Full Text]
    
20. Nickerson PA, Yang F. Calcium distribution in aortic smooth muscle cells of deoxycorticosterone-hypertensive rats: a quantitative cytochemical study. J Submicrosc Cytol Pathol. 1988; 20: 317–324.[Medline] [Order article via Infotrieve]
    
21. Wuorela H. The effect of high calcium intake on intracellular free [Ca²⁺] and Na⁺-H⁺ exchange in DOC-NaCl-hypertensive rats. Pharmacol Toxicol. 1992; 71: 376–382.[Medline] [Order article via Infotrieve]
    
22. Haller H, Thiede M, Lenz T, Ludersdorf M, Harwig S, Distler A, Philipp T. Intracellular free calcium and ionized plasma calcium during mineralocorticoid-induced blood pressure increase in man. J Hypertens Suppl. 1985; 3 (suppl 3): S41–S43.[Medline] [Order article via Infotrieve]
    
23. Li W, Zheng T, Altura BT, Altura BM. Antioxidants prevent elevation in [Ca²⁺]_i induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg²⁺ deficiency-induced vasospasm and stroke. Brain Res Bull. 2000; 52: 151–154.[CrossRef][Medline] [Order article via Infotrieve]
    
24. Weglicki WB, Mak IT, Phillips PM. Blockade of cardiac inflammation in Mg²⁺ deficiency by substance P receptor inhibition. Circ Res. 1994; 74: 1009–1013.[Abstract/Free Full Text]
    
25. Weglicki WB, Mak IT, Stafford RE, Dickens BF, Cassidy MM, Phillips TM. Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: effects of substance P-receptor inhibition. Mol Cell Biochem. 1994; 130: 103–109.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Malpuech-Brugere C, Kuryszko J, Nowacki W, Rock E, Rayssiguier Y, Mazur A. Early morphological and immunological alterations in the spleen during magnesium deficiency in the rat. Magnes Res. 1998; 11: 161–169.[Medline] [Order article via Infotrieve]
    
27. Bussière FI, Gueux E, Rock E, Mazur A, Rayssiguier Y. Protective effect of calcium deficiency on the inflammatory response in magnesium-deficient rats. Eur J Nutr. 2002; 41: 197–202.[CrossRef][Medline] [Order article via Infotrieve]
    
28. Bussière FI, Zimowska W, Gueux E, Rayssiguier Y, Mazur A. Stress protein expression cDNA array study supports activation of neutrophils during acute magnesium deficiency in rats. Magnes Res. 2002; 15: 37–42.[Medline] [Order article via Infotrieve]
    
29. Fukuda K, Baba A, Kuchii M, Nakamura Y, Nishio I, Masuyama Y. Increased concentration of calcium channel and intracellular free calcium in the acute phase of deoxycorticosterone acetate salt hypertension. J Hypertens Suppl. 1988; 6: S261–S263.[Medline] [Order article via Infotrieve]
    
30. Bénitah JP, Vassort G. Aldosterone upregulates Ca²⁺ current in adult rat cardiomyocytes. Circ Res. 1999; 85: 1139–1145.[Abstract/Free Full Text]
    
31. Lesouhaitier O, Chiappe A, Rossier MF. Aldosterone increases T-type calcium currents in human adrenocarcinoma (H295R) cells by inducing channel expression. Endocrinology. 2001; 142: 4320–4330.[Abstract/Free Full Text]
    
32. Rossier MF, Lesouhaitier O, Perrier E, Bockhorn L, Chiappe A, Lalevée N. Aldosterone regulation of T-type calcium channels. J Steroid Biochem Mol Biol. 2003; 85: 383–388.[CrossRef][Medline] [Order article via Infotrieve]
    
33. Lijnen P, Fagard R, Petrov V. Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells. J Cardiovasc Pharmacol. 1999; 33: 595–604.[CrossRef][Medline] [Order article via Infotrieve]
    
34. Nebe B, Holzhausen C, Rychly J, Urbaszek W. Impaired mechanisms of leukocyte adhesion in vitro by the calcium channel antagonist mibefradil. Cardiovasc Drugs Ther. 2002; 16: 183–193.[CrossRef][Medline] [Order article via Infotrieve]
    
35. Ramires FJA, Sun Y, Weber KT. Myocardial fibrosis associated with aldosterone or angiotensin II administration: attenuation by calcium channel blockade. J Mol Cell Cardiol. 1998; 30: 475–483.[CrossRef][Medline] [Order article via Infotrieve]
    
36. Baylis C, Qiu C, Engels K. Comparison of L-type and mixed L- and T-type calcium channel blockers on kidney injury caused by deoxycorticosterone-salt hypertension in rats. Am J Kidney Dis. 2001; 38: 1292–1297.[Medline] [Order article via Infotrieve]
    
37. Dworkin LD, Levin RI, Benstein JA, Parker M, Ullian ME, Kim Y, Feiner HD. Effects of nifedipine and enalapril on glomerular injury in rats with deoxycorticosterone-salt hypertension. Am J Physiol. 1990; 259: F598–F604.[Medline] [Order article via Infotrieve]
    
38. Randriamampita C, Trautmann A. Ca²⁺ signals and T lymphocytes: "New mechanisms and functions in Ca²⁺ signalling." Biol Cell. 2004; 96: 69–78.[CrossRef][Medline] [Order article via Infotrieve]
    
39. Ohkuma S, Katsura M, Higo A, Shirotani K, Hara A, Tarumi C, Ohgi T. Peroxynitrite affects Ca²⁺ influx through voltage-dependent calcium channels. J Neurochem. 2001; 76: 341–350.[CrossRef][Medline] [Order article via Infotrieve]
    
40. Bussière FI, Gueux E, Rock E, Girardeau JP, Tridon A, Mazur A, Rayssiguier Y. Increased phagocytosis and production of reactive oxygen species by neutrophils during magnesium deficiency in rats and inhibition by high magnesium concentration. Br J Nutr. 2002; 87: 107–113.[CrossRef][Medline] [Order article via Infotrieve]
    
41. Berthon N, Laurant P, Hayoz D, Fellmann D, Brunner HR, Berthelot A. Magnesium supplementation and deoxycorticosterone acetate-salt hypertension: effect on arterial mechanical properties and on activity of endothelin-1. Can J Physiol Pharmacol. 2002; 80: 553–561.[CrossRef][Medline] [Order article via Infotrieve]
    
42. Berthon N, Laurant P, Fellmann D, Berthelot A. Effect of magnesium on mRNA expression and production of endothelin-1 in DOCA-salt hypertensive rats. J Cardiovasc Pharmacol. 2003; 42: 24–31.[CrossRef][Medline] [Order article via Infotrieve]
    
43. Chang JM, Lai YH, Tsai JH. Interaction between extracellular calcium and endothelin-1 influences parathyroid hormone secretion from bovine parathyroid cells through the increase in intracellular calcium. Miner Electrolyte Metab. 1997; 23: 113–120.[Medline] [Order article via Infotrieve]
    
44. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, Wittes W, for the Randomized Aldactone Evaluation Study Investigators. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med. 1999; 341: 709–717.[Abstract/Free Full Text]
    
45. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, Gatlin M. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med. 2003; 348: 1309–1321.[Abstract/Free Full Text]
    
46. Diaz-Velez CR, Garcia-Castineiras S, Mendoza-Ramos E, Hernandez-Lopez E. Increased malondialdehyde in peripheral blood of patients with congestive heart failure. Am Heart J. 1996; 131: 146–152.[CrossRef][Medline] [Order article via Infotrieve]
    
47. Chen L, Zang Y, Bai B, Zhu M, Zhao B, Hou J, Xin W. Electron spin resonance determination and superoxide dismutase activity in polymorphonuclear leukocytes in congestive heart failure. Can J Cardiol. 1992; 8: 756–760.[Medline] [Order article via Infotrieve]
    
48. Tsutsui H, Ide T, Hayashidani S, Suematsu N, Shiomi T, Wen J, Nakamura K, Ichikawa K, Utsumi H, Takeshita A. Enhanced generation of reactive oxygen species in the limb skeletal muscles from a murine infarct model of heart failure. Circulation. 2001; 104: 134–136.[Abstract/Free Full Text]
    
49. Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze TH, Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res. 2001; 89: 279–286.[Abstract/Free Full Text]
    
50. Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K, Colucci WS. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol. 2002; 34: 379–388.[CrossRef][Medline] [Order article via Infotrieve]
    
51. Anker SD, Chua TP, Ponikowski P, Harrington D, Swan JW, Kox WJ, Poole-Wilson PA, Coats AJS. Hormonal changes and catabolic/anabolic imbalance in chronic heart failure and their importance for cardiac cachexia. Circulation. 1997; 96: 526–534.[Abstract/Free Full Text]
    

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