Inhaled nitric oxide improves gas exchange, decreases pulmonary vascular lability, and reduces pulmonary inflammation. We hypothesized that the use of inhaled nitric oxide would decrease the incidence of chronic lung disease and death in premature infants with the respiratory distress syndrome.

Rationale : 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. Methods and 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.

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. To determine whether the hypoxia-induced increases in mitochondrial ROS act to trigger calcium increases, we measured changes in cytosolic calcium ([Ca ²⁺ ] _i ) using fura 2-AM (fluorescence at 340/380 nm) during perfusion with hypoxic media (P o ₂ 12 mm Hg). Hypoxia caused an increase in fura 2 fluorescence, indicating an increase in [Ca ²⁺ ] _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 [Ca ²⁺ ] _i increases. To test whether mitochondrial H ₂ O ₂ is required to trigger [Ca ²⁺ ] _i increases, catalase was overexpressed in PA myocytes with the use of a recombinant adenovirus. Catalase overexpression attenuated hypoxia-induced increases in [Ca ²⁺ ] _i , suggesting that H ₂ O ₂ acts upstream from calcium increases during hypoxia. These results support the conclusion that mitochondria function as O ₂ 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.

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.

Chronic lung disease and severe intraventricular hemorrhage or periventricular leukomalacia in premature infants are associated with abnormal neurodevelopmental outcomes. In a previous randomized, controlled, single-center trial of premature infants with the respiratory distress syndrome, inhaled nitric oxide decreased the risk of death or chronic lung disease as well as severe intraventricular hemorrhage and periventricular leukomalacia. We hypothesized that infants treated with inhaled nitric oxide would also have improved neurodevelopmental outcomes.

Rationale: The role of reactive oxygen species (ROS) signaling in the O2 sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). Measurements and Main Results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca2+]i. In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca2+]i of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca2+]i that cause acute hypoxic pulmonary vasoconstriction.