Hypoxia activates NADPH oxidase to increase ROSi and Ca2+i through mitochondrial ROS–PKCε signaling axis in pulmonary artery smooth muscle cells

摘要

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, p22^phox, p47^phox, and p67^phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47^phox protein to the plasma membrane in pulmonary, but not mesenteric arteries. The Nox inhibitor apocynin and p47^phox gene deletion attenuated hypoxic increase in intracellular reactive oxygen species and Ca²⁺ concentration ([ROS]i and [Ca²⁺]i) as well as contraction in mouse PASMCs, and abolished hypoxic activation of Nox in pulmonary arteries. The conventionalovel protein kinase C (PKC) inhibitor chelerythrine, specific PKCε translocation peptide inhibitor and PKCε gene deletion, but not the conventional PKC inhibitor GÖ6976, prevented hypoxic increase in Nox activity in pulmonary arteries and in [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 hypoxic activation of Nox, whereas Gpx1 gene deletion had an 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 mitochondrial ROS–PKCε signaling axis, providing a positive feedback mechanism to contribute to hypoxic increase in [ROS]i and [Ca²⁺]i as well as contraction in PASMCs.