Abstract-We hypothesized that mitochondria function as the O 2 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. Key Words: reactive oxygen species Ⅲ hypoxia Ⅲ redox signaling Ⅲ pulmonary circulation Ⅲ oxidants H ypoxic pulmonary vasoconstriction (HPV) diverts blood flow away from the lung during fetal development and optimizes lung gas exchange after birth by enhancing the matching of blood flow and ventilation. Excised lungs retain the HPV response, 1-6 as do rings of the pulmonary artery (PA) 7,8 even when they are denuded of endothelium. 9,10 Even isolated PA myocytes contract during hypoxia, 11 indicating that an O 2 sensor is intrinsic to those cells.Although HPV has been well characterized, the underlying mechanism of O 2 sensing is not established. Mitochondria have long been known to generate reactive oxygen species (ROS), 12 although these oxidants have classically been viewed as toxic byproducts of the electron transport pathway, possibly contributing to the effects of aging. 13 More recently, mitochondrial ROS have been implicated as intracellular signaling agents. We previously reported that mitochondria increase ROS generation during hypoxia and that site-specific inhibition of electron transport could attenuate hypoxiainduced ROS generation. 14,15 In isolated buffered-perfused rat lungs and isolated PA myocytes, we found that inhibition of the mitochondrial electron transport chain (ETC) upstream from complex III attenuated HPV. 16 By contrast, inhibition downstream from complex III either had no effect or augmented HPV. 16 The likely explanation for these findings is that O 2 -dependent ROS production occurs within complex III. In the Q cycle, a free radical (ubisemiquinone) is normally generated during the electron transport process. This radical can potentially donate its unpaired electron to O 2 , thereby generating superoxide. Our model suggests that the process of ROS generation from that site is amplified during hypoxia.In support of this model, we found that antioxidants selectively abolish the HPV response in isolated lungs. 16 However, that study did not test whether the calcium increases during hypoxia required ROS production. Accordingly, the present study sought to determine whether oxidant production from the mitochondria is responsible for triggering calcium increases and therefore myocyte contraction during HPV. This hypothesis was tested by measuring the effects of site-specific mitochondrial inhibitors and antioxidants on calcium signaling in primary cultured PA myocytes under hypoxic conditions.

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 hypoxiainduced increases in cytosolic calcium ([Ca 2ϩ ] i ), assessed by the Ca 2ϩ -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 2ϩ ] i . Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca 2ϩ ] i , whereas mitochondrial matrix-targeted Mn-SOD (SOD-II) augmented [Ca 2ϩ ] i . The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca 2ϩ ] i , whereas cyanide augmented the increase in [Ca 2ϩ ] i . Finally, simultaneous measurement of ROS and Ca 2ϩ 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 2ϩ ] i by augmenting ROS signaling from the mitochondria. Key Words: hypoxic pulmonary vasoconstriction Ⅲ reactive oxygen species Ⅲ redox signaling Ⅲ antioxidants Ⅲ fluorescence resonance energy transfer A lthough hypoxic pulmonary vasoconstriction (HPV) was first described by von Euler and Liljestrand in 1946, 1 the underlying mechanism by which vascular cells detect the decrease in O 2 tension has not been established. Hypoxia activates an O 2 sensor that triggers contraction of pulmonary artery smooth muscle cells (PASMCs) through an increase in cytosolic calcium ([Ca 2ϩ ] i ) via release of Ca 2ϩ from the sarcoplasmic reticulum and/or entry of extracellular Ca 2ϩ through voltage-, receptor-, or store-operated channels in the sarcolemma. [2][3][4][5][6][7][8] However, the signaling pathways that couple the O 2 sensor to the increases in [Ca 2ϩ ] i have not been established.Mitochondria have long been considered putative sites of oxygen sensing because they consume O 2 and therefore represent the intracellular site with the lowest oxygen tension.Two opposing views have emerged regarding the nature of the...