Abstract
Hyperaldosteronism is associated with impaired endothelium-dependent vascular reactivity owing to increased reactive oxygen species and decreased bioavailable nitric oxide (NO(.)); however, the effects of aldosterone on vasodilatory signaling pathways in vascular smooth muscle cells (VSMC) remain unknown. Soluble guanylyl cyclase (GC) is a heterodimer that is activated by NO(.) to convert cytosolic GTP to cGMP, a second messenger required for normal VSMC relaxation. Here, we show that aldosterone (10(-9)-10(-7) mol/liter) diminishes GC activity by activating NADPH oxidase in bovine aortic VSMC to increase reactive oxygen species levels and induce oxidative posttranslational modification(s) of Cys-122, a beta(1)-subunit cysteinyl residue demonstrated previously to modulate NO(.) sensing by GC. In VSMC treated with aldosterone, Western immunoblotting detected evidence of GC beta(1)-subunit disulfide bonding, whereas mass spectrometry analysis of a homologous peptide containing the Cys-122-bearing sequence exposed to conditions of increased oxidant stress confirmed cysteinyl sulfinic acid (m/z 435), sulfonic acid (m/z 443), and disulfide (m/z 836) bond formation. The functional effect of these modifications was examined by transfecting COS-7 cells with wild-type GC or mutant GC containing an alanine substitution at Cys-122 (C122A). Exposure to aldosterone or hydrogen peroxide (H(2)O(2)) significantly decreased cGMP levels in cells expressing wild-type GC. In contrast, aldosterone or H(2)O(2) did not influence cGMP levels in cells expressing the mutant C122A GC, confirming that oxidative modification of Cys-122 specifically impairs GC activity. These findings demonstrate that pathophysiologically relevant concentrations of aldosterone increase oxidant stress to convert GC to an NO(.)-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in VSMC.
Highlights
Elevated levels of the mineralocorticoid hormone aldosterone are associated with impaired vascular reactivity in patients with an aldosterone-producing adenoma, hypertension, and congestive heart failure that is remediable following surgical resection of the tumor or treatment with a mineralocorticoid receptor antagonist [1,2,3,4,5]
Aldosterone or H2O2 did not influence cGMP levels in cells expressing the mutant C122A guanylyl cyclase (GC), confirming that oxidative modification of Cys-122 impairs GC activity. These findings demonstrate that pathophysiologically relevant concentrations of aldosterone increase oxidant stress to convert GC to an NO1⁄7-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in vascular smooth muscle cells (VSMC)
We hypothesized that aldosterone-mediated ROS formation promotes oxidative posttranslational modification of GC and conversion of the enzyme to an NO1⁄7-insensitive state, thereby disrupting a key pathway essential for normal VSMC relaxation
Summary
Cell Culture and Treatments—Bovine aortic VSMC (Genlantis) were grown to confluence using phenol-free Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin, at 37 °C, 5% CO2. NO1⁄7 Metabolites—Nitrite (NO2Ϫ) and nitrate (NO3Ϫ) were measured from cell culture medium containing 2% fetal bovine serum and L-arginine (1 mmol/liter)(Sigma) by 2,3-diaminonaphthalene fluorescence (Cayman). Cells were collected and centrifuged at 1,500 ϫ g for 10 min at 4 °C, cGMP formation was measured by immunoassay according to the manufacturer’s instructions (Cayman), and bicinchoninic acid (Bio-Rad) was used for protein determination. After a 1-h incubation at 25 °C, proteins were precipitated with acetone, resuspended in 50 l of non-reducing SDS electrophoresis buffer, size-fractionated electrophoretically using SDS-PAGE, and transferred to a polyvinylidene fluoride membrane [23]. COS-7 cells, which do not express endogenous GC, were plated in 6-well tissue culture dishes and transfected with 5 g of DNA for 4 h with Lipofectamine 2000TM (Invitrogen) [24] After this time, medium was replaced with full growth media, and experiments were performed after 48 h. Comparison between groups was performed by Student’s paired two-tailed t test, and p Ͻ 0.05 is considered significant
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