Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells
Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells
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DOI：
10.1074/jbc.m809460200
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发表时间：
2009-03-20
影响因子：
4.8
通讯作者：
Leopold, Jane A.
中科院分区：
文献类型：
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作者：
Maron, Bradley A.;Zhang, Ying-Yi;Leopold, Jane A.
醛固酮增多症与内皮依赖性血管反应性受损有关，这是由于活性氧增加和生物可利用的一氧化氮（NO中心点）减少;然而，醛固酮对血管平滑肌细胞（VSMC）中血管舒张信号通路的影响仍不清楚。可溶性鸟苷酸环化酶（GC）是一种异源二聚体，被NO中心点激活，将胞浆GTP转化为cGMP，cGMP是正常VSMC松弛所需的第二信使。在此，我们发现醛固酮（10（-9）-10（-7）mol/L）通过激活牛主动脉VSMC中的NADPH氧化酶来降低GC活性，从而增加活性氧水平并诱导Cys-122的氧化翻译后修饰，Cys-122是一种β（1）亚基半胱氨酰残基，以前被证明可调节GC对NO中心点的感知。在用醛固酮处理的VSMC中，Western免疫印迹检测到GC β（1）亚基二硫键的证据，而暴露于增加的氧化应激条件下的含有Cys-122-轴承序列的同源肽的质谱分析证实了半胱氨酰亚磺酸（m/z 435），磺酸（m/z 443）和二硫键（m/z 836）的形成。通过用野生型GC或在Cys-122（C122 A）处含有丙氨酸取代的突变型GC转染COS-7细胞来检查这些修饰的功能效果。暴露于醛固酮或过氧化氢（H2 O2）可显着降低表达野生型GC的细胞中的cGMP水平。相比之下，醛固酮或H2 O2不影响表达突变型C122 A GC的细胞中的cGMP水平，证实Cys-122的氧化修饰特异性地损害GC活性。这些研究结果表明，病理生理相关浓度的醛固酮增加氧化应激转换GC的NO中心点不敏感的状态，导致正常的血管舒张信号通路在VSMC的破坏。
Hyperaldosteronism is associated with impaired endothelium-dependent vascular reactivity owing to increased reactive oxygen species and decreased bioavailable nitric oxide (NO center dot); 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 center dot 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 center dot 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 (H2O2) significantly decreased cGMP levels in cells expressing wild-type GC. In contrast, aldosterone or H2O2 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 center dot-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in VSMC.