Abstract 357: Aldosterone Inactivates the Endothelin-B Receptor via a Thiol Redox Switch to Modulate Pulmonary Arterial Hypertension
Hyperaldosteronism (ALDO) induces a vasculopathy in systemic blood vessels by increasing reactive oxygen species (ROS) that decrease nitric oxide (NO) levels. In pulmonary artery endothelial cells (PAECs), the endothelin-B receptor (ETB) activates endothelial NO synthase (eNOS) to generate NO. The effect of ALDO-induced ROS on ETB-NO signaling in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that in PAH, ALDO increases ROS levels in PAECs, which oxidatively modify the regulatory ETB cysteinyl thiol Cys405 to impair ETB activation of eNOS and decrease NO levels. To test this hypothesis, human PAECs were treated with vehicle (V) or ALDO (10-7 mol/l) for 24 h and hydrogen peroxide (H2O2) levels were assessed by Amplex Red assay. Compared to V-treated cells, ALDO increased significantly H2O2 levels (65.4 ± 1.6 vs. 100.6 ± 3.5 μM/μg protein, p<0.05). In cells exposed to endothelin-1 (ET-1)(10 nM) to stimulate ETB signaling, ALDO decreased eNOS activity by 59% (p<0.05) and nitrite (NO2-) formation by 60% (p<0.05) without affecting eNOS protein levels. ALDO-induced ROS led to the oxidative formation of two disulfide bonds in ETB as detected by PEG-maleimide-labeled immunoblotting. To confirm that oxidative modification of Cys405 influenced ETB-dependent NO generation, COS7 cells were transfected with wild type (WT)-eNOS and WT-ETB or mutant ETB DNA containing an alanine substitution at position 405 (C405A-ETB), and cells were exposed to V or H2O2 (200 μM) for 60 min. We observed that H2O2 decreased ETB-mediated NO2- formation by 78% (p<0.02) in WT-ETB-transfected cells; in C405A-ETB-transfected cells, H2O2 decreased NO2- levels by only 45%. Next, we assessed the effect of ALDO on NO signaling in PAH in vivo. In monocrotaline (MCT)-treated rats with PAH, plasma ALDO was increased by 406% compared to V-treated rats (p<0.02). ALDO antagonism with spironolactone (25 mg/kg/d) increased NO2- levels in lung homogenates of MCT-treated rats by 50% (p<0.05) and decreased significantly pulmonary artery systolic pressure by cardiac catheterization (89 ± 5 vs. 69 ± 5 mm Hg, p<0.04). These data suggest that ALDO/the regulatory redox switch of ETB, Cys405, may represent novel therapeutic target(s) to restore ETB-NO signaling in patients with PAH.
- © 2012 by American Heart Association, Inc.