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.

Rationale: The mobilization of bone marrow (BM) progenitor cells (PCs) is largely governed by interactionsbetween stromal cell-derived factor (SDF)-1 and CXC chemokine receptor (CXCR)4. Ischemic injury disrupts the SDF-1-CXCR4 interaction and releases BM PCs into the peripheral circulation, where the mobilized cells are recruited to the injured tissue and contribute to vessel growth. BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, but the other components of the SDF-1-CXCR4 signaling pathway are largely unknown. c-kit, a membrane-bound tyrosine kinase and the receptor for stem cell factor, has also been shown to play a critical role in BM PC mobilization and ischemic tissue repair.Objective: To investigate the functional interaction between SDF-1-CXCR4 signaling and c-kit activity in BM PC mobilization. Key Words: CXCR4 Ⅲ c-kit Ⅲ stem cells Ⅲ bone marrow Ⅲ mobilization T he mobilization or release of progenitor cells (PCs) from the bone marrow (BM) to the peripheral blood (PB) is highly regulated and occurs both under normal conditions and in response to stress. 1,2 PB PCs have an essential role in blood cell formation and homeostasis 3 and in the response of various tissues to noxious insults. Thus, therapies that enhance PC mobilization are being investigated as novel strategies for promoting tissue repair. 4,5 Ample evidence suggests that PCs are retained in the BM by interactions between the CXC chemokine stromal cell-derived factor (SDF)-1 and CXC chemokine receptor (CXCR)4. 6 -10 CXCR4 is expressed by BM mononuclear cells (MNCs), and SDF-1 is expressed by osteoblasts, endothelial cells, and a subset of reticular cells scattered throughout the BM. 7 Ischemic injury disrupts the SDF-1-CXCR4 interaction and releases BM PCs into the peripheral circulation; then, the mobilized cells are recruited to Original received March 25, 2010; revision received August 25, 2010; accepted August 27, 2010. In August 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days. Methods and ResultsFrom the injured tissue and contribute to vessel growth. 11,12 BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, 13-15 but the downstream components of the SDF-1-CXCR4 signaling pathway are largely unknown. Like CXCR4, c-kit is expressed predominantly in BM PCs, and the ligand for c-kit, stem cell factor (SCF), is constitutively produced by BM endothelial cells and fibroblasts. 16 c-kit is a class III receptor tyrosine kinase, and administration of a c-kit-neutralizing antibody (ACK2) to wild-type (WT) mice released BM cells to the peripheral circulation and enhanced the engraftment of intravenously injected BM cells. 17 On the other hand, PC mobilization is markedly blunted in c-kit W/W-V mutant mice, which are defective in c-kit kinase activity but have normal levels of c-kit expression and SCF binding at the cell surface. 18 -21 Furthermore, the kinetics of BM PC mobilization induced by the c-kit...

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