Abstract 11683: Aldosterone Oxidizes a Thiol Redox Switch in the Endothelin-B Receptor to Decrease Pulmonary Endothelial Nitric Oxide Levels and Promote Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension
Hyperaldosteronism (ALDO) increases endothelial hydrogen peroxide (H2O2) levels to decrease nitric oxide (NO) levels in the systemic vasculature; however, the effect of ALDO on NO synthesis in the pulmonary circulation is unknown. In pulmonary artery endothelial cells (PAECs), stimulation of the endothelin-B receptor (ETB) activates nitric oxide synthase (eNOS) to increase NO. We hypothesized that ALDO disrupts ETB-eNOS signaling by oxidizing a cysteinyl thiol redox switch in ETB to decrease NO synthesis and promote pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). To test this hypothesis, human PAECs were treated with vehicle control (V) or ALDO (10-7 mol/L) for 24 h and H2O2 levels were measured by Amplex Red assay. Compared to V, ALDO increased H2O2 levels by 57% (p<0.05). ALDO also decreased ETB-dependent nitrite (NO2-) formation by 60% (p<0.05). We next determined if ALDO-induced H2O2 oxidizes ETB cysteinyl thiols Cys402, Cys403, and Cys405 (Cys402/3/5) located in the eNOS-activating region of ETB. Cells treated with V or ALDO were immunoprecipitated with an antibody specific to the region of ETB containing Cys402/3/5. Anti-sulfenic acid (RSOH) immunoblotting showed that ALDO increased ETB-SOH levels 6.2-fold (p<0.05). To confirm that Cys402/3/5 oxidation influenced ETB-dependent NO synthesis, COS7 cells were transfected with wild type (WT)-eNOS and WT-ETB or mutant ETB DNA containing an alanine substitution at positions 402/3/5 (C402/3/5A-ETB) and cells were exposed to V or H2O2 (200 μM) for 60 min. In WT-ETB-transfected cells, H2O2 decreased ETB-mediated NO2- by 70%; in C402/3/5A-transfected cells, H2O2 decreased NO2- by only 17% (p<0.05). In rats with monocrotaline-induced PAH, ALDO levels were increased in lung tissue 1.7-fold (p<0.04). Compared to V-treated rats with PAH, ALDO antagonism with spironolactone (25 mg/kg/d) increased NO2- levels by 50% (p<0.05) in lung tissue and decreased the number of muscularized pulmonary arterioles detected by α-actin immunohistochemistry (77.7 ± 6.3 vs. 59.7 ± 0.4 vessels/20 hpf, p<0.05). These data suggest that ALDO/ETB Cys402/3/5 are thiol redox switch(es) that may represent novel therapeutic target(s) for restoring NO levels and attenuating vascular remodeling in PAH.
- © 2012 by American Heart Association, Inc.