Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through the mitochondrial ROS-PKCɛ signaling axis in pulmonary artery smooth muscle cells

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22phox, p47phox, and p67phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47phox protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47phox gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca2+ ([ROS]i and [Ca2+]i), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCɛ translocation peptide inhibitor, and PKCɛ gene deletion, but not the conventional PKC inhibitor GÖ6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS]i in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H2O2 removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H2O2 increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS]i through the mitochondrial ROS-PKCɛ signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS]i and [Ca2+]i as well as contraction in PASMCs.