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

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