Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells

Ryugo Sakurada, Keiichi Odagiri, Akio Hakamata, Chiaki Kamiya, Jiazhang Wei, Hiroshi Watanabe
International Journal of Molecular Sciences 2019 April 2, 20 (7)

BACKGROUND: Previous studies demonstrated that calcium/calmodulin (Ca2+ /CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+ /CaM-dependent pathway.

METHODS: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays.

RESULTS: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+ -free or 1 mM of extracellular Ca2+ concentration modified Tyrode's solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+ /CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5.

CONCLUSIONS: BK-induced NOX-derived ROS production was mediated via a Ca2+ /CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.

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