Aldosterone increases oxidant stress to impair guanylyl cyclase activity by cysteinyl thiol oxidation in vascular smooth muscle cells.
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.
Maron
Bradley A
BA
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Zhang
Ying-Yi
YY
Handy
Diane E
DE
Beuve
Annie
A
Tang
Shiow-Shih
SS
Loscalzo
Joseph
J
Leopold
Jane A
JA
D000450:Aldosterone / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006162:Guanylate Cyclase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006929:Hyperaldosteronism / Q000201:enzymology / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D051381:Rats
D004220:Disulfides; D016877:Oxidants; D009569:Nitric Oxide; D000450:Aldosterone; D006861:Hydrogen Peroxide; D006162:Guanylate Cyclase