title: Increases in mitochondrial reactive oxygen species trigger hypoxia-induced calcium responses in pulmonary artery smooth muscle cells.
abstract: 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+)](i)), 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+)](i). Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca(2+)](i), whereas mitochondrial matrix-targeted Mn-SOD (SOD-II) augmented [Ca(2+)](i). The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca(2+)](i), whereas cyanide augmented the increase in [Ca(2+)](i). 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+)](i) by augmenting ROS signaling from the mitochondria.
authors.affiliation: Department of Pediatrics, Division of Neonatology, Northwestern University, Chicago, IL 60611, USA. g-waypa@northwestern.edu
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Waypa
authors.forename: Robert
authors.initials: R
authors.lastname: Guzy
authors.forename: Paul T
authors.initials: PT
authors.lastname: Mungai
authors.forename: Mathew M
authors.initials: MM
authors.lastname: Mack
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Marks
authors.forename: Michael W
authors.initials: MW
authors.lastname: Roe
authors.forename: Paul T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D015687:Cell Hypoxia; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005979:Glutathione Peroxidase / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000502:physiology; D010084:Oxidation-Reduction; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D014661:Vasoconstriction
chemical_list: D017382:Reactive Oxygen Species; D005979:Glutathione Peroxidase; D002118:Calcium