Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.
BACKGROUND
Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation.


OBJECTIVE
This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells.


RESULTS
We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs.


CONCLUSIONS
These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.
Waypa
Gregory B
GB
Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Marks
Jeremy D
JD
Guzy
Robert
R
Mungai
Paul T
PT
Schriewer
Jacqueline
J
Dokic
Danijela
D
Schumacker
Paul T
PT
D000818:Animals; D002374:Catalase / Q000096:biosynthesis / Q000235:genetics; D002451:Cell Compartmentation; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010084:Oxidation-Reduction; D011651:Pulmonary Artery / Q000166:cytology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012077:Renal Artery / Q000166:cytology; D013347:Subcellular Fractions / Q000378:metabolism*; D014661:Vasoconstriction / Q000502:physiology
D017077:Culture Media, Conditioned; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002374:Catalase