2] Detection of oxygen-sensing properties of mitochondria

Abstract

The chapter describes two techniques described to detect the role of mitochondria in regulating the hypoxic generation of reactive oxygen species (ROS) and activation of hypoxia inducible factor 1 ( HIF-1). In the first method, the generation of ROS and the activation of HIF-1 are examined in cells depleted of mtDNA (p° cells). The second method examines the generation of ROS and the activation of HIF-1 in cells treated with mitochondrial inhibitors. Accompanying these methods are protocols for detecting nuclear HIF-1 α protein levels by immunoblotting and detecting intracellular ROS by the oxidation of 2', 7'-dichlorofluorescin (DCFH). In theory, the same principles and methods can be applied in examining a different hypoxic response, such as activation of kinases or other transcription factors. Currently, there are two redox-dependent models to explain oxygen sensing in mammalian cells. Another model assumes that an oxygen sensor located in the cytosol detects decreases in oxygen tension by decreasing the generation of ROS. In an alternative model, it is proposed that mitochondria detect decreases in oxygen concentration and respond by increasing the generation of ROS that are required and sufficient to activate HIF-1. At the cellular level, one of the most crucial responses to hypoxia is the activation of the transcription factor HIF-1. HIF-1 is a heterodimer of two basic helix loop-helix/PAS proteins: HIF-1 α and the aryl hydrocarbon nuclear translocator (ARNT or HIF-1 β).