Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes.
We hypothesized that mitochondria function as the O2 sensors underlying hypoxic pulmonary vasoconstriction by releasing reactive oxygen species (ROS) from complex III of the electron transport chain (ETC). We have previously found that antioxidants or inhibition of the proximal region of the ETC attenuates hypoxic pulmonary vasoconstriction in rat lungs and blocks hypoxia-induced contraction of isolated pulmonary arterial (PA) myocytes. To determine whether the hypoxia-induced increases in mitochondrial ROS act to trigger calcium increases, we measured changes in cytosolic calcium ([Ca2+]i) using fura 2-AM (fluorescence at 340/380 nm) during perfusion with hypoxic media (PO2 12 mm Hg). Hypoxia caused an increase in fura 2 fluorescence, indicating an increase in [Ca2+]i. In superfused PA myocytes, diphenyleneiodonium, rotenone, and myxothiazol, which inhibit the proximal region of the ETC, attenuated hypoxia-induced calcium increases. Antimycin A and cyanide, which inhibit the distal region of the ETC, failed to abolish hypoxia-induced [Ca2+]i increases. To test whether mitochondrial H2O2 is required to trigger [Ca2+]i increases, catalase was overexpressed in PA myocytes with the use of a recombinant adenovirus. Catalase overexpression attenuated hypoxia-induced increases in [Ca2+]i, suggesting that H2O2 acts upstream from calcium increases during hypoxia. These results support the conclusion that mitochondria function as O2 sensors during hypoxia and demonstrate that ROS generated in the proximal region of the ETC act as second messengers to trigger calcium increases in PA myocytes during acute hypoxia.
Waypa
Gregory B
GB
Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, Ill 60637, USA.
Marks
Jeremy D
JD
Mack
Mathew M
MM
Boriboun
Chan
C
Mungai
Paul T
PT
Schumacker
Paul T
PT
D000818:Animals; D020013:Calcium Signaling; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003486:Cyanides / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005420:Flavoproteins / Q000037:antagonists & inhibitors; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015290:Second Messenger Systems
D003486:Cyanides; D005420:Flavoproteins; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C007517:diphenyleneiodonium