Increases in Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Calcium Responses in Pulmonary Artery Smooth Muscle Cells

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Abstract
Mitochondria have been implicated as a potential site of O2 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 ([Ca2+]i), assessed by the Ca2+-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca2+]i. Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca2+]i, whereas mitochondrial matrix–targeted Mn-SOD (SOD-II) augmented [Ca2+]i. The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca2+]i, whereas cyanide augmented the increase in [Ca2+]i. Finally, simultaneous measurement of ROS and Ca2+ 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 [Ca2+]i by augmenting ROS signaling from the mitochondria.

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