Hypoxia Triggers Subcellular Compartmental Redox Signaling in Vascular Smooth Muscle Cells

Abstract

Key Words: mitochondria Ⅲ reactive oxygen species Ⅲ ratiometric fluorescent protein sensor Ⅲ hypoxic pulmonary vasoconstriction S mall pulmonary arteries constrict in response to a decrease in alveolar oxygen tension through a physiological response termed hypoxic pulmonary vasoconstriction (HPV). Despite intensive study since the first description of HPV in 1946, the mechanism of O 2 sensing remains unclear. Mitochondria have been considered a putative site of cellular oxygen sensing because they consume O 2 and therefore represent the intracellular site with the lowest oxygen tension. Mitochondria generate superoxide and hydrogen peroxide during cellular respiration, so changes in the production of reactive oxygen species (ROS) during hypoxia could potentially signal changes in O 2 tension and trigger cellular responses. However, whether hypoxia increases or decreases ROS generation during HPV remains unclear.Using chemiluminescence in whole perfused lungs and endothelium-denuded rings of distal pulmonary arteries, Michelakis, Archer, and Weir and colleagues detected decreases in ROS generation during hypoxia. [1][2][3][4] Similarly, in cultured pulmonary arterial pulmonary artery smooth muscle cells (PASMCs), Mehta et al detected a decrease in ROS generation as determined by dihydrodichlorofluorescein diacetate (DCF), dihydroethidium, and Amplex red. 5 By contrast, using cultured PASMCs, we found that hypoxia triggers an increase in ROS generation. 6 -8 Our studies used the ROS-sensitive indicator dichlorofluorescein (DCF), mitochondrial inhibitors, enzymatic and chemical antioxidants and mitochondria-deficient ( 0 ) cells. Other investigators have detected similar increases in ROS generation during hypoxia by using lucigenin-derived chemiluminescence and electron paramagnetic resonance spectrometry in small pulmonary arteries, as well as DCF in PASMCs. 8,9 Attempts to resolve the question of whether ROS levels increase or decrease during hypoxia have been hindered by the technical limitations of the tools used to assess oxidant stress. Although lucigenin can detect ROS, lucigenin itself is capable of producing superoxide in cells. 9 Furthermore,

Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation

“…In contrast, oxidation in the mitochondrial matrix decreased from 46.1 6 2.9% during normoxia to 21.7 6 2.3% after 30 minutes of hypoxia ( Figure 1C). These results in mouse PASMC are consistent with previous results obtained in rat PASMC (10).…”

“…Some of the results of these studies have been previously reported in the form of abstracts (30,31).…”

“…To assess ROS signaling in subcellular compartments of cultured wild-type mouse PASMC during hypoxia, we infected cells with adenoviruses harboring DNA to express roGFP targeted to the cytosol, mitochondrial matrix, or mitochondrial intermembrane space (IMS-roGFP) as we have done previously (10). We used roGFP to assess protein thiol oxidation; to our knowledge this reporter does not participate in signal transduction.…”

“…This response is recapitulated in cultured PA smooth muscle cells (PASMC), indicating that the oxygen-sensing mechanism underlying HPV is intrinsic to the PASMC (1-12). Our previous work has implicated increases in reactive oxygen species (ROS) signaling during hypoxia (9,10,12). Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9,(11)(12)(13)(14)(15)(16)(17)(18).…”

“…Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9,(11)(12)(13)(14)(15)(16)(17)(18). We subsequently assessed ROS signaling in hypoxic PASMC using roGFP, a thiol-containing, redox-sensitive reporter (19)(20)(21)(22)(23) targeted to compartments within mitochondria or the cytosol (10). Unlike other methods (24)(25)(26), this targeted approach permitted the differentiation of hypoxia-induced ROS changes between mitochondrial subcompartments.…”