Induction of hypoxia inducible factor 2α (HIF‐2α) in an immortalized adrenomedullary chromaffin cell line requires mitochondrial‐derived reactive oxygen species

Hypoxia‐induced catecholamine release from adrenomedullary chromaffin cells (AMC) plays a critical role in the ability of the neonate to adapt to air‐breathing life, and occurs via a direct non‐neurogenic mechanism. One important set of regulators in such O2‐sensitive cells is the HIF family of transcription factors (e.g. HIF‐1α, HIF‐2α, HIF‐3α) which are stabilized during hypoxia and can regulate a host of genes that are essential for survival. However, there is controversy as to whether or not HIF stabilization requires mitochondrial‐derived reactive oxygen species (ROS). To address this, we are using an immortalized rat adrenomedullary chromaffin (MAH) cell line which was previously shown to be O2‐sensitive and shares several properties with neonatal AMC (Fearon et al., J. Physiol., 545:807–818, 2002). In MAH cells, HIF‐2α induction occurs during 1 hr exposure to chronic hypoxia (2% O2) and remains elevated over at least 24 hrs. However, the induction of HIF‐2α after a 4 hr hypoxic exposure decreases in the presence of the antioxidant, ascorbic acid (200 μM), suggesting the involvement of ROS. The relevant ROS appears to be mitochondrial‐derived since rotenone (0.5 μg/ml), a mitochondrial complex I inhibitor, also decreased HIF‐2α induction during hypoxia. This effect of rotenone was reversed by the addition of the dimethyl (R)‐(+)‐methyl succinate (5 mM), a permeable analog of the complex II substrate, succinate. To investigate further the proposed role of mitochondrial ROS in the induction of HIF by chronic hypoxia, we have successfully generated mitochondrial‐depleted (ρ0) MAH cells which will be used in future studies. Supported by a grant from the Heart and Stroke Foundation of Ontario.