Abstract
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.
Highlights
Reactive oxygen species (ROS) have classically been considered a harmful byproduct of oxidant metabolism, as excessive reactive oxygen species (ROS) leads to the progression of vascular senescence, atherosclerosis, plaque instability, and endothelial dysfunction in the vascular system [1,2,3]
Considering that sarcoplasmic reticulum Ca2+ release could activate the CaM/CaM-dependent protein kinase II (CaMKII) pathway, as we previously demonstrated in [15], we aimed to determine whether Ca2+ mobilization from the endoplasmic reticulum (ER), but not store-operated Ca2+ entry (SOCE), was required to regulate nicotinamide adenine dinucleotide phosphate oxidases (NOX)-derived ROS via Ca2+/CaM-dependent pathways in porcine aortic endothelial cells (PAECs)
Pretreatment with 1,2-Bis (2 aminophenoxy) ethane-N,N,N’,N-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM, 100 μM) in Ca2+-free modified Tyrode’s solution abolished BK-induced Ca2+ responses (Figure 1B). These results indicated that 1 μM of BK introduced SOCE, and BAPTA/AM eliminated the effect of BK on ER Ca2+ mobilization
Summary
Reactive oxygen species (ROS) have classically been considered a harmful byproduct of oxidant metabolism, as excessive ROS leads to the progression of vascular senescence, atherosclerosis, plaque instability, and endothelial dysfunction in the vascular system [1,2,3]. The representative enzymatic sources of ROS in endothelial cells are: (1) nitric oxide synthase, (2) the mitochondrial respiratory chain, (3) xanthine dehydrogenase, and (4) nicotinamide adenine dinucleotide phosphate oxidases (NOX). NOX are a known contributor of redox-sensitive signaling pathways that lead to endothelial dysfunction [10,11]. Little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. We investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. Conclusions: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents
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