| Summary: | Background: Previous studies demonstrated that calcium/calmodulin (Ca<sup>2+</sup>/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca<sup>2+</sup> level consists of two components: Ca<sup>2+</sup> mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca<sup>2+</sup> entry. However, little is known about which component of Ca<sup>2+</sup> 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 Ca<sup>2+</sup>/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 Ca<sup>2+</sup>-free or 1 mM of extracellular Ca<sup>2+</sup> concentration modified Tyrode’s solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca<sup>2+</sup> by BAPTA/AM or the depletion of ER Ca<sup>2+</sup> contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca<sup>2+</sup>/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 Ca<sup>2+</sup>/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca<sup>2+</sup> contents.
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