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(Circulation Research. 2006;99:970.)
© 2006 American Heart Association, Inc.

Cellular Biology

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

Gregory B. Waypa, Robert Guzy, Paul T. Mungai, Mathew M. Mack, Jeremy D. Marks, Michael W. Roe, Paul T. Schumacker

From the Department of Pediatrics (G.B.W., R.G., P.T.M., P.T.S.), Division of Neonatology, Northwestern University, Chicago; Department of Pediatrics (J.D.M.), Section of Neonatology, and Department of Medicine (M.W.R., M.M.M.), Pulmonary and Critical Care Section (M.M.M.), The University of Chicago, Ill.

Correspondence to Gregory B. Waypa, Department of Pediatrics, Northwestern University, Ward Bldg 12-189, 303 E Chicago Ave, Chicago, IL 60611. E-mail g-waypa{at}northwestern.edu

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.

Key Words: hypoxic pulmonary vasoconstriction • reactive oxygen species • redox signaling • antioxidants • fluorescence resonance energy transfer

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