Effects of Hypoxia on intracellular H2S and Polysulfides: Implications in O2 Sensing

The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generation of reactive oxygen species (ROS). However, we recently found that methods commonly employed to measure ROS cannot distinguish between ROS and reactive sulfur species (RSS) and that RSS and ROS have similar effects on intracellular redox status. Our previous studies suggest that metabolism of hydrogen sulfide (H 2 S) is the O 2 sensor, as H 2 S mimics hypoxia in numerous systems and H 2 S metabolism is inversely coupled to O 2 availability at physiological levels. In the present study we measure intracellular production of H 2 S and H 2 S n using 7‐Azido‐4‐Methylcoumarin (AzMC) and 3,6‐DI(O‐Thiosalicyl) (SSP4), respectively in HEK‐293 cells and bovine pulmonary artery smooth muscle cells (BPASMC) under both normoxic (21% O 2 ) and hypoxic (0.5% O 2 ) conditions over a 48 hr period. In addition, we attempted to augment H 2 S and H 2 S n production via the known intracellular pathways through addition of substrates (to increase production) and inhibitors (to decrease production). We found that hypoxia increased AzMC fluorescence by 50%, but did not affect SSP4 fluorescence. In normoxia, AzMC fluorescence did not change, whereas SSP4 fluorescence increased two‐fold. These results suggest that hypoxia increases intracellular H 2 S concentration and concomitantly prevents further the increase in H 2 Sn concentration. Intracellular H 2 S and H 2 Sn was not affected by either cysteine or cystine (10 and 100 uM). Both propargylglycine (10 mM), an inhibitor of cystathionine ã‐lyase and aspartate (1 mM) an inhibitor of cysteine aminotransferase reduced hypoxia stimulated AzMC fluorescence in HEK‐293 cells. Aminooxyacetate, an inhibitor of cystathionione â‐synthase, greatly increased AzMC fluorescence in HEK‐293 cells in normoxia and hypoxia. These are the first measurements of oxygen‐dependent changes in intracellular H 2 S and H 2 Sn in living cells and they support our hypothesis that RSS are directly coupled to O 2 sensing. Support or Funding Information NSF Grant IOS‐144‐6310 (KRO), NSF GRFP DGE‐131‐3583 (ERD)