Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species...

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.

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

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.

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.