Waypa Gregory B, Guzy Robert, Mungai Paul T, Mack Mathew M, Marks Jeremy D, Roe Michael W, Schumacker Paul T
Department of Pediatrics, Division of Neonatology, Northwestern University, Chicago, IL 60611, USA.
Circ Res. 2006 Oct 27;99(9):970-8. doi: 10.1161/01.RES.0000247068.75808.3f. Epub 2006 Sep 28.
Mitochondria have been implicated as a potential site of O(2) sensing underlying hypoxic pulmonary vasoconstriction (HPV), but 2 disparate models have been proposed to explain their reaction to hypoxia. One model proposes that hypoxia-induced increases in mitochondrial reactive oxygen species (ROS) generation activate HPV through an oxidant-signaling pathway, whereas the other proposes that HPV is a result of decreased oxidant signaling. In an attempt to resolve this debate, we use a novel, ratiometric, redox-sensitive fluorescence resonance energy transfer (HSP-FRET) probe, in concert with measurements of reduced/oxidized glutathione (GSH/GSSG), to assess cytosolic redox responses in cultured pulmonary artery smooth muscle cells (PASMCs). Superfusion of PASMCs with hypoxic media increases the HSP-FRET ratio and decreases GSH/GSSG, indicating an increase in oxidant stress. The antioxidants pyrrolidinedithiocarbamate and N-acetyl-l-cysteine attenuated this response, as well as the hypoxia-induced increases in cytosolic calcium (Ca(2+)), assessed by the Ca(2+)-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and Ca(2+). Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and Ca(2+), whereas mitochondrial matrix-targeted Mn-SOD (SOD-II) augmented Ca(2+). The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and Ca(2+), whereas cyanide augmented the increase in Ca(2+). Finally, simultaneous measurement of ROS and Ca(2+) signaling in the same cell revealed that the initial increase in these 2 signals could not be distinguished temporally. These results demonstrate that hypoxia triggers increases in PASMC Ca(2+) by augmenting ROS signaling from the mitochondria.
线粒体被认为是低氧性肺血管收缩（HPV）潜在的氧传感位点，但已提出两种不同的模型来解释其对缺氧的反应。一种模型认为，缺氧诱导线粒体活性氧（ROS）生成增加，通过氧化信号通路激活HPV，而另一种模型则认为HPV是氧化信号减少的结果。为了解决这一争论，我们使用一种新型的、比率型、对氧化还原敏感的荧光共振能量转移（HSP-FRET）探针，并结合还原型/氧化型谷胱甘肽（GSH/GSSG）的测量，来评估培养的肺动脉平滑肌细胞（PASMCs）中的胞质氧化还原反应。用缺氧培养基灌注PASMCs会增加HSP-FRET比率并降低GSH/GSSG，表明氧化应激增加。抗氧化剂吡咯烷二硫代氨基甲酸盐和N-乙酰-L-半胱氨酸减弱了这种反应，以及通过钙敏感FRET传感器YC2.3评估的缺氧诱导的胞质钙（Ca(2+)）增加。腺病毒介导的谷胱甘肽过氧化物酶或胞质或线粒体过氧化氢酶的过表达减弱了缺氧诱导的ROS信号和Ca(2+)的增加。腺病毒介导的胞质铜锌超氧化物歧化酶（SOD-I）的过表达对缺氧诱导的ROS信号和Ca(2+)的增加没有影响，而线粒体基质靶向的锰超氧化物歧化酶（SOD-II）增强了Ca(2+)。线粒体抑制剂粘噻唑减弱了缺氧诱导的ROS信号和Ca(2+)的变化，而氰化物增强了Ca(2+)的增加。最后，在同一细胞中同时测量ROS和Ca(2+)信号发现，这两种信号的初始增加在时间上无法区分。这些结果表明，缺氧通过增强线粒体的ROS信号来触发PASMC Ca(2+)的增加。
