A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells.
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.
Thompson
R J
RJ
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1. rthom@interchange.ubc.ca
Buttigieg
J
J
Zhang
M
M
Nurse
C A
CA
D000111:Acetylcysteine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000166:cytology*; D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects*; D004558:Electric Stimulation / Q000379:methods; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008163:Luminescent Measurements / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D016877:Oxidants / Q000494:pharmacology*; D018408:Patch-Clamp Techniques / Q000379:methods; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000031:analogs & derivatives* / Q000494:pharmacology
D000975:Antioxidants; D016877:Oxidants; D017382:Reactive Oxygen Species; D012402:Rotenone; C028404:rotenolone; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D011188:Potassium; D000111:Acetylcysteine