Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells

Waypa, G. B.; Marks, J. D.; Guzy, R.; Mungai, P. T.; Schriewer, J.; Dokic, D.; Schumacker, P. T.

Circ Res. 2009 Dec 19; 106(3):526-35

Abstract

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.

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