Aldosterone inactivates the endothelin-B receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO(·)) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ET(B)), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species generation and decreasing NO(·) levels. We hypothesized that aldosterone modulates PAH by disrupting ET(B)-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress.

Methods and results: In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO(·) metabolites in the absence of left-sided heart failure. In human pulmonary artery endothelial cells, endothelin-1 increased aldosterone levels via peroxisome proliferator-activated receptor gamma coactivator-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased reactive oxygen species production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ET(B) to decrease endothelin-1-stimulated eNOS activity. Substitution of ET(B)-Cys405 with alanine improved ET(B)-dependent NO(·) synthesis under conditions of oxidant stress, confirming that Cys405 is a redox-sensitive thiol that is necessary for ET(B)-eNOS signaling. In human pulmonary artery endothelial cells, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated reactive oxygen species generation and restored ET(B)-dependent NO(·) production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in 2 animal models of PAH in vivo.

Conclusions: Our findings demonstrate that aldosterone modulates an ET(B) cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO(·) and promote PAH.

Figure 1

Elevated levels of ET-1 are associated with hyperaldosteronism in PAH. (a,b) Levels of ET-1 (n=4–6) and (c,d) aldosterone (ALDO) (n=4–8) were measured in plasma and lung tissue homogenates of Sprague-Dawley rats 25 days following treatment with vehicle control (V) or monocrotaline (MCT) (50 mg/kg). Horizontal line represents the median for each condition.

Figure 2

Aldosterone promotes PAH in vivo. Sprague-Dawley rats were treated with vehicle control (V) or monocrotaline (MCT) (50 mg/kg) and randomized immediately to V or spironolactone (SP) (25 mg/kg/d) for 25 days (n=6 rats per condition). The contribution of aldosterone to PAH was assessed by (a) right heart catheterization to measure pulmonary artery (assumed to be equivalent to right ventricular) systolic pressure (PASP); echocardiography to assess changes in (b) pulmonary artery acceleration time (PAAT); and, (c) right ventricular (RV) free-wall thickness. Horizontal line represents the mean for each condition.

Figure 3

Spironolactone increases pulmonary vascular NO^• levels and attenuates pulmonary vascular remodeling in PAH. (a) The effect of spironolactone (SP)(25 mg/kg/d) on pulmonary vascular NO^• levels in PAH was assessed by measuring nitrite (NO₂⁻) in lung tissue homogenates from Sprague-Dawley rats treated with vehicle control (V) or monocrotaline (MCT) (50 mg/kg) (n=4). (b) Tissue sections were stained with anti-smooth muscle cell α-actin antibody and the number of muscularized distal pulmonary arterioles (red arrows) was counted in 20 consecutive fields per section (100x magnification). Compared to V-treated rats with PAH, spironolactone decreased significantly the number of α-actin-stained muscularized distal pulmonary arterioles (76.0 [64–95] vs. 59.5 [59–61] muscularized pulmonary arterioles/20 high powered fields, p<0.005, n=5). (c) Gomori’s trichrome stain was performed on paraffin-embedded lung sections and perivascular collagen deposition in pulmonary arterioles measuring 20–50 μm located distal to terminal bronchioles (400x magnification) was measured. Compared to V-treated rats with PAH, spironolactone decreased perivascular collagen deposition by 77% (p<0.001, n=4–5 rats per condition). Representative photomicrographs are shown.

Figure 4

The effect of mineralocorticoid receptor antagonism on reversal or prevention of adverse cardiopulmonary hemodynamics in two models of experimental PAH. (a) In a reversal study, Sprague-Dawley rats were randomized to receive vehicle control (V) or spironolactone (SP) (25 mg/kg/d) 14 days following the administration of V or monocrotaline (MCT) (50 mg/kg), and cardiopulmonary hemodynamics were assessed by cardiac catheterization 10 days later. *p<0.02 vs. MCT, n=6 rats per condition; **p<0.04 vs. V, n=4 rats per condition. Data are presented as mean ± S.E. (b) In a prevention study, Sprague-Dawley rats were injected with SU-5416 and exposed to chronic hypoxia for 21 days. Immediately following exposure to hypoxia, rats were randomized to receive standard chow or eplerenone (0.6 gm/1 gm chow) until completion of the study. The effect of eplerenone on pulmonary artery systolic pressure (PASP) was assessed by cardiac catheterization (n=5 rats per condition). HR, heart rate; CI, cardiac index; LVEDP, left ventricular end-diastolic pressure; PVRi, pulmonary vascular resistance index; SVRi, systemic vascular resistance index.

Figure 5

ET-1 stimulates PGC-1α-dependent association of SF with CYP11B2 to increase aldosterone levels. (a) The effect of ET-1 on PGC-1α expression was assessed by Western analysis (n=4). (b) Co-immunoprecipitation experiments demonstrated that incubation of HPAECs with ET-1 (10 nM) for 24 h induced the association of PGC-1α with steroidogenesis factor-1 (SF) (n=3). (c) Chromatin immunoprecipitation (n=3) of cell lysates using antibodies to PGC-1α, SF, and immunoglobulin-G (IgG) as a negative control was followed by PCR amplification of the proximal region of the CYP11B2 promoter region containing the gonadotrope-specific element. (d) The functional effect of PGC-1α stimulation on aldosterone production was assessed in cells treated with the selective PGC-1α agonist pioglitazone (50 μM) for 24 h (n=4), or with ET-1 (10 nM) or angiotensin II (ANG)(10 μM) for 24 h as positive controls. PGC-1α, PPAR-γ co-activator-1α; arb. units, arbitrary units; IP, immunoprecipitation, IB; immunoblot. Data are presented as mean ± S.E.M. Representative blots are shown.

Figure 6

Aldosterone decreases ET_B-dependent synthesis of NO^•. (a) HPAECs were exposed to vehicle (V) or aldosterone (ALDO) (10⁻⁷ mol/L) for 24 h in the presence or absence of spironolactone (SP) (10 μM) and NO₂⁻ formation was assessed. Prior to analysis, cells were exposed to ET-1 (10 nM) for 10 min to stimulate ET_B signaling (n=4). (b) The effect of ALDO on ET_B-dependent activation of eNOS was determined (n=4). c.p.m., counts per minute. (c) The effect of ALDO on ET_B-dependent NO^• generation was assessed by measuring total NO^• metabolite levels (NOx: NO₂⁻ + NO₃⁻) (n=3). (d) HPAECs were exposed to V, hydrogen peroxide (H₂O₂) (200 μM) for 20 min, or ALDO (10⁻⁷ mol/L) for 24 h to assess changes to the redox status and de novo disulfide bond formation by ET_B cysteinyl thiols. For each disulfide formed, a 20-kDa shift in band location of the reduced ET_B protein occurs on the Western blot using an antibody specific to the region of ET_B containing Cys405 (n=4). Cyss, disulfide bond. A representative blot is shown. (e) The region of ET_B containing Cys405 was immunoprecipitated from cells treated with V or ALDO (10⁻⁷ mol/L) for 24 h and immunoblotting was performed to detect differences in protein sulfenic acid levels (R-SOH) (n=3). IP, immunoprecipitation, IB; immunoblot. Data are presented as mean ± S.E.M. Representative blots are shown.

Figure 7

Oxidation of Cys405 impairs ET_B-dependent NO^• generation. (a) COS-7 cells were transiently transected with wild type (WT)-eNOS and WT-ET_B or mutant ET_B DNA containing a substitution of alanine for cysteine at position 405 (C405A-ET_B) and protein expression was confirmed. No Tx, untransfected. (b) Disulfide bond formation was assessed by Western immunoblotting of PEG-conjugated maleimide-labeled cell extracts exposed to H₂O₂ (200 μmol/L for 20 min). Compared to WT-ET_B-transfected cells, in which H₂O₂ (200 μmol/L for 20 min) induced the formation of 1 or 2 disulfide bonds, C405A-ET_B was resistant to disulfide bond formation (n=4) Cyss, disulfide bond. (c) COS-7 cells expressing WT-eNOS and WT-ET_B or C405A-ET_B were exposed to vehicle (V) control or hydrogen peroxide (H₂O₂) (200 μmol/L) for 60 min. After that time, the cell culture medium was replaced and cells were treated with ET-1 (10 nM) for 10 min. and nitrite (NO₂⁻) levels were measured (n=4). Data are presented as mean ± S.E.M. Representative blots are shown.

Figure 8

A proposed mechanism by which hyperaldosteronism decreases pulmonary endothelial eNOS activation and NO^• generation in PAH. Hyperaldosteronism (ALDO) in pulmonary arterial hypertension (PAH) may occur via i) endothelin-1 (ET-1)-mediated activation of PPARγ coactivator-1α (PGC-1α)/steroidogenesis factor-1 (SF) to increase CYP11B2 (aldosterone synthase) gene transcription in HPAECs, and/or ii) upregulation of adrenal ALDO synthesis via ET-1 and/or overactivation of the renin-angiotensin pathway. Stimulation of the mineralocorticoid receptor (MR) in HPAECs by ALDO activates NADPH oxidase type 4 (NOX4) to increase levels of hydrogen peroxide (H₂O₂), which, in turn, oxidatively modifies redox sensitive, functional cysteinyl thiol(s) in the ET_B receptor (Cys405) to impair ET_B-dependent activation of eNOS and decrease synthesis of nitric oxide (NO^•). eNOS, endothelial nitric oxide synthase; R-SO_XH, higher oxidative intermediaries of cysteine.