Hypoxia upregulates the expression of the cardioprotective peptide adrenomedullin in cardiomyocytes. We characterized this pathway in murine HL-1 cardiomyocytes. Inhibition of mitochondrial complexes I, III, and IV largely, but not completely, reduced hypoxic adrenomedullin mRNA increase in gas-impernicable Culture plates. Complex III inhibition was also effective in permeable culture plates, so that this effect is unlikely due to intracellular oxygen redistribution, whereas complex I blockade was ineffective in permeable plates. Complex II does not participate in this effect, as shown by chemical and siRNA inactivation. ROS scavenging by nitroblue tetrazolium and general flavoprotein inhibition by diplienyleniodonium nearly abrogated the hypoxic adrenoinedullin mRNA increase. Thus, ROS production by flavoproteins is crucial for hypoxic upregulation of adrenoiliedullin mRNA in murine HL-1 cardiomyocytes. These ROS originate both from the mitochondrial complex III and from additional,. presumably extramitochondrial, Sources. Mitochondrial oxygen consumption appears to have impact on oxygen availability at these extramitochondrial sensors. (c) 2006 Elsevier Inc. All rights reserved.