Endoplasmic reticulum Ca(2+) release causes Rieske iron–sulfur protein-mediated mitochondrial ROS generation in pulmonary artery smooth muscle cells

Mitochondrial reactive oxygen species (ROS) cause Ca(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) in pulmonary artery smooth muscle cells (PASMCs), playing an essential role in hypoxic pulmonary vasoconstriction (HPV). Here we tested a novel hypothesis that hypoxia-induced RyR-mediated Ca(2+) release may, in turn, promote mitochondrial ROS generation contributing to hypoxic cellular responses in PASMCs. Our data reveal that application of caffeine to elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) by activating RyRs results in a significant increase in ROS production in cytosol and mitochondria of PASMCs. Norepinephrine to increase [Ca(2+)](i) due to the opening of inositol 1,4,5-triphosphate receptors (IP3Rs) produces similar effects. Exogenous Ca(2+) significantly increases mitochondrial-derived ROS generation as well. Ru360 also inhibits the hypoxic ROS production. The RyR antagonist tetracaine or RyR2 gene knockout (KO) suppresses hypoxia-induced responses as well. Inhibition of mitochondrial Ca(2+) uptake with Ru360 eliminates N- and Ca(2+)-induced responses. RISP KD abolishes the hypoxia-induced ROS production in mitochondria of PASMCs. Rieske iron–sulfur protein (RISP) gene knockdown (KD) blocks caffeine- or NE-induced ROS production. Taken together, these findings have further demonstrated that ER Ca(2+) release causes mitochondrial Ca(2+) uptake and RISP-mediated ROS production; this novel local ER/mitochondrion communication-elicited, Ca(2+)-mediated, RISP-dependent ROS production may play a significant role in hypoxic cellular responses in PASMCs.