Department of Pediatrics, Northwestern University, Chicago, IL 60611, USA.
Antioxid Redox Signal. 2012 Aug 1;17(3):460-70. doi: 10.1089/ars.2011.4184. Epub 2012 Mar 8.
Oxygen is a pulmonary vasodilator, but data suggest high O(2) concentrations impede that response. We previously reported 24 h of 100% O(2) increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5.
Thirty minutes of hyperoxia increased mitochondrial ROS versus normoxia (30.3±1.7% vs. 21.1±2.8%), but had no effect on cytosolic ROS, measured by roGFP, a ratiometric protein thiol redox sensor. Hyperoxia increased PDE5 activity (220±39%) and decreased cGMP responsiveness to NO (37±17%). Mitochondrial catalase overexpression attenuated hyperoxia-induced mitochondrial roGFP oxidation, compared to FPASMC infected with empty adenoviral vector (50±3% of control) or mitochondrial superoxide dismutase. MitoTEMPO, mitochondrial catalase, and DT-3, a cGMP-dependent protein kinase I alpha inhibitor, decreased PDE5 activity (32±13%, 26±21%, and 63±10% of control, respectively), and restored cGMP responsiveness to NO (147±16%,172±29%, and 189±43% of control, respectively). C57Bl6 mice exposed to 90%-100% O(2) for 45 min±mechanical ventilation had increased PA PDE5 activity (206±39% and 235±75%, respectively).
This is the first description that hyperoxia induces ROS in the mitochondrial matrix prior to the cytosol. Our results indicate that short hyperoxia exposures can produce significant changes in critical cellular signaling pathways.
These results indicate that mitochondrial matrix oxidant signals generated during hyperoxia, specifically H(2)O(2), activate PDE5 in a cGMP-dependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells.
氧气是一种肺血管扩张剂，但数据表明高氧浓度会阻碍这种反应。我们之前报道过，24 小时 100%的氧气增加了胎儿肺动脉平滑肌细胞（FPASMC）和通气新生羔羊的磷酸二酯酶 5（PDE5）活性。PDE5 降解环鸟苷酸（cGMP）并抑制一氧化氮（NO）介导的 cGMP 依赖性血管舒张。我们试图确定高氧引发活性氧（ROS）产生并调节 PDE5 的机制。
与常氧相比，30 分钟的高氧增加了线粒体 ROS（30.3±1.7% vs. 21.1±2.8%），但对细胞浆 ROS 没有影响，这是通过 ratiometric 蛋白硫醇氧化还原传感器 roGFP 测量的。高氧增加了 PDE5 活性（220±39%），并降低了 cGMP 对 NO 的反应性（37±17%）。与感染空腺病毒载体的 FPASMC（对照组的 50±3%）或线粒体超氧化物歧化酶相比，线粒体过氧化氢酶过表达减轻了高氧诱导的线粒体 roGFP 氧化。MitoTEMPO、线粒体过氧化氢酶和 cGMP 依赖性蛋白激酶 Iα抑制剂 DT-3 降低了 PDE5 活性（分别为对照组的 32±13%、26±21%和 63±10%），并恢复了 cGMP 对 NO 的反应性（分别为对照组的 147±16%、172±29%和 189±43%）。暴露于 90%-100%氧气 45 分钟±机械通气的 C57Bl6 小鼠肺动脉 PDE5 活性增加（分别为 206±39%和 235±75%）。
这是第一个描述高氧在细胞质之前在线粒体基质中诱导 ROS 的描述。我们的结果表明，短暂的高氧暴露会导致关键细胞信号通路发生显著变化。
这些结果表明，高氧期间产生的线粒体基质氧化剂信号，特别是 H2O2，以 cGMP 依赖性蛋白激酶依赖的方式在肺血管平滑肌细胞中激活 PDE5。
