O2‐sensing in immortalized rat adrenomedullary chromaffin (MAH) cells: Role of mitochondria and ROS signaling

Hypoxia‐induced catecholamine (CA) release from neonatal adrenomedullary chromaffin cells (AMC) is critical during the adaptation of the newborn to extrauterine life and is facilitated by inhibition of O2−sensitive K+ channels. Using a surrogate model based on a v‐myc immortalized adrenal chromaffin cell line (i.e. MAH cells), which shares several O2 sensing properties with neonatal AMC, we tested the hypothesis that mitochondrial function and changes in reactive oxygen species (ROS) were involved in O2 sensing. In these studies we used whole‐cell recordings to monitor outward K+ currents, the chemiluminescence probe luminol to detect ROS levels, luciferin‐luciferase chemiluminescence to detect extracellular ATP (co‐released with CA), and ratiometric fura‐2 measurements to monitor changes in intracellular Ca2+. In MAH cells, hypoxia (PO2 ~ 20 mmHg) caused an inhibition of outward K+ current, an increase in intracellular Ca2+ and ATP release (that was cadmium sensitive), and a decrease in ROS levels during ~3 min exposure. Rotenone (1 μM), a blocker of mitochondrial complex I, decreased ROS and increased ATP release in MAH cells; interestingly, the combined effects of rotenone and hypoxia were non‐additive. The ROS scavenger N‐acetyl‐cysteine (50 μM) caused the expected decrease in ROS, and moreover, inhibited outward K+ current and stimulated ATP release. Interestingly, mitochondrial‐deficient or ρ0 MAH cells (generated by the ethidium bromide technique) lacked hypoxic sensitivity, though K+ currents appeared normal and secretory responses to high [K+] remained intact. These data suggest that functional mitochondria and ROS signaling play critical roles in the hypoxic sensitivity of adrenal chromaffin cells. Supported by Heart and Stroke Foundation of Ontario.