In the perinatal period, adrenomedullary chromaffin cells (AMC) directly sense PO2 and secrete
catecholamines during hypoxic stress, and this response is lost in juvenile ( approximately 2 week-old) chromaffin cells following postnatal innervation. Here we tested the hypothesis that a
rotenone-sensitive O2-sensor and ROS are involved in the hypoxic response of AMC cultured from neonatal and juvenile rats. In whole-cell recordings,
hypoxia (PO2=5-15 mm Hg) inhibited outward current in neonatal AMC; this response was reversed by exogenous H2O2 and mimicked and occluded by intracellular
catalase (1000 units/ml), as well as the
antioxidants,
N-acetyl-L-cysteine (NAC; 50 microM) and
Trolox (200 microM). Acute
hypoxia decreased ROS levels and stimulated
ATP secretion in these cells, as measured by
luminol and
luciferin-
luciferase chemiluminescence, respectively. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only
rotenone, a complex I blocker, mimicked and occluded the effects of
hypoxia on outward current, cellular ROS, and
ATP secretion.
Succinate donors, which act as complex II substrates, reversed the effects of
hypoxia and
rotenone in neonatal AMC. In contrast, in
hypoxia-insensitive juvenile AMC, neither NAC nor
rotenone stimulated
ATP secretion though they both caused a decrease in ROS levels. We propose that O2-sensing by neonatal AMC is mediated by decreased ROS generation via a
rotenone-sensitive site that is coupled to outward current inhibition and secretion. Interestingly, juvenile AMC display at least two modifications, i.e. an uncoupling of the O2-sensor from ROS regulation, and an apparent insensitivity of outward current to decreased ROS.