Abstract
Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.
Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.
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References
Kaelin WG Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30(4):393–402. https://doi.org/10.1016/j.molcel.2008.04.009
López-Barneo J, Ortega-Sáenz P, González-Rodríguez P, Fernández-Agüera MC, Macías D, Pardal R, Gao L (2016) Oxygen-sensing by arterial chemoreceptors: mechanisms and medical translation. Mol Asp Med 47–48:90–108. https://doi.org/10.1016/j.mam.2015.12.002
Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 95(20):11715–11720. https://doi.org/10.1073/pnas.95.20.11715
Quintero M, Colombo SL, Godfrey A, Moncada S (2006) Mitochondria as signaling organelles in the vascular endothelium. Proc Natl Acad Sci U S A 103(14):5379–5384. https://doi.org/10.1073/pnas.0601026103
Chua YL, Dufour E, Dassa EP, Rustin P, Jacobs HT, Taylor CT, Hagen T (2010) Stabilization of hypoxia-inducible factor-1alpha protein in hypoxia occurs independently of mitochondrial reactive oxygen species production. J Biol Chem 285(41):31277–31284. https://doi.org/10.1074/jbc.M110.158485
Fernández-Agüera MC, Gao L, González-Rodríguez P, Pintado CO, Arias-Mayenco I, García-Flores P, García-Pergañeda A, Pascual A, Ortega-Sáenz P, López-Barneo J (2015) Oxygen sensing by arterial chemoreceptors depends on mitochondrial complex I signaling. Cell Metab 22(5):825–837. https://doi.org/10.1016/j.cmet.2015.09.004
Gao L, González-Rodríguez P, Ortega-Sáenz P, López-Barneo J (2017) Redox signaling in acute oxygen sensing. Redox Biol 12:908–915. https://doi.org/10.1016/j.redox.2017.04.033
Izquierdo-Álvarez A, Ramos E, Villanueva J, Hernansanz-Agustín P, Fernández-Rodríguez R, Tello D, Carrascal M, Martínez-Ruiz A (2012) Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia. J Proteome 75(17):5449–5462. https://doi.org/10.1016/j.jprot.2012.06.035
Hernansanz-Agustín P, Izquierdo-Álvarez A, Sánchez-Gómez FJ, Ramos E, Villa-Piña T, Lamas S, Bogdanova A, Martínez-Ruiz A (2014) Acute hypoxia produces a superoxide burst in cells. Free Radic Biol Med. https://doi.org/10.1016/j.freeradbiomed.2014.03.011
Hernansanz-Agustín P, Ramos E, Navarro E, Parada E, Sánchez-López N, Peláez-Aguado L, Cabrera-García JD, Tello D, Buendia I, Marina A, Egea J, López MG, Bogdanova A, Martínez-Ruiz A (2017) Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia. Redox Biol 12:1040–1051. https://doi.org/10.1016/j.redox.2017.04.025
Hernansanz-Agustín P, Choya-Foces C, Carregal-Romero S, Ramos E, Oliva T, Villa-Piña T, Moreno L, Izquierdo-Álvarez A, Cabrera-García JD, Cortés A, Lechuga-Vieco AV, Jadiya P, Navarro E, Parada E, Palomino-Antolón A, Tello D, Acín-Pérez R, Rodríguez-Aguilera JC, Navas P, Cogolludo Á, López-Montero I, Martínez-del-Pozo Á, Egea J, López MG, Elord JW, Bogdanova A, Enríquez JA, Martínez-Ruiz A (2019) Mitochondrial Na+ import controls oxidative phosphorylation and hypoxic redox signalling. bioRxiv 385690. https://doi.org/10.1101/385690
Acknowledgments
Research in our lab is supported by grants PI15/00107 and RTI2018-094203-B-I00 from the Spanish Government (partially funded by the European Union ERDF) and by a grant from the Fundación Domingo Martínez.
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Hernansanz-Agustín, P., Choya-Foces, C., Martínez-Ruiz, A. (2021). Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy. In: Espada, J. (eds) Reactive Oxygen Species. Methods in Molecular Biology, vol 2202. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0896-8_3
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DOI: https://doi.org/10.1007/978-1-0716-0896-8_3
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