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

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.

NADPH oxidase in the vasculature: Expression, regulation and signalling pathways; role in normal cardiovascular physiology and its dysregulation in hypertension

“…The primary site of acute regulation of NOX5 is an N-terminal cytosolic domain containing four EF hands that bind free Ca 2+ , as well as Ca 2+ -calmodulin binding domains, that collectively relieve oxidase auto-inhibition [235,236]. As a result, NOX5-derived ROS production is enhanced by stimuli that raise intracellular Ca 2+ concentration ([Ca 2+ ]i) in endothelium and VSM, such as thrombin, Ang II, ET-1, U46619 and bradykinin, acting both via voltage-dependent extracellular Ca 2+ influx and sarcoplasmic reticulum Ca 2+ -induced Ca 2+ release [55,66,232,237]. NOX5 is therefore essentially constitutively active but with a 'basal' activity that is dependent on [Ca 2+ ]i [66].…”

“…As in VSM, NOX isoforms 1, 2, 4 and 5 are all expressed in the vascular endothelium[53,54,215,234,[413][414][415] and are likely sources of the ROS contributing to or influencing these relaxation responses. Indeed, acetylcholine, histamine, bradykinin and flow all induce NOX-derived ROS production in vascular tissues and cultured endothelial cells[237,402,405,408,414,[416][417][418][418][419][420][421] (Table1). Vascular relaxation responses in isolated arteries and cerebral blood flow are improved by non-selective NOX inhibitors…”

The Roles of Superoxide on At-Level Spinal Cord Injury Pain in Rats

“…It is well known that an increase in calcium ion release from cytoplasmic calcium stores activates calcium-dependent protein kinase [ 36 ], and that activated CamKII maintains its active form throughout autophosphorylation [ 37 , 38 , 39 ]. These results suggest that ROS-mediated calcium levels are involved in the activation of CamKII, rather than the influx of calcium ions, which is mediated by the ionotropic glutamate receptor.…”

Skeletal muscle DNMT3A plays a necessary role in endurance exercise by regulating oxidative capacity of red muscles

“…The resultant potassium influx into the matrix lowers the mitochondrial membrane potential, which causes mitochondrial swelling, opening of permeability transition pores, and elevated ROS production [23][24][25]. In addition, NOX-derived ROS causes leakage of Ca2+ from the sarcoplasmic reticulum or entry of extracellular Ca2+, resulting in mitochondrial Ca2+ overload and mitochondrial ROS emission, which ultimately results in muscle fatigue and dysfunction [13,[17][18][19][26][27][28].…”

“…The resultant potassium influx into the matrix lowers the mitochondrial membrane potential, which causes mitochondrial swelling, opening of permeability transition pores, and elevated ROS production [23][24][25]. In addition, NOX-derived ROS causes leakage of Ca2+ from the sarcoplasmic reticulum or entry of extracellular Ca2+, resulting in mitochondrial Ca2+ overload and mitochondrial ROS emission, which ultimately results in muscle fatigue and dysfunction [13,[17][18][19][26][27][28].Exercise significantly alters the DNA methylation profile of skeletal muscle [29][30][31][32][33][34][35][36][37]. DNA methylation, a reversible epigenetic mark that usually occurs on a cytosine residue followed by a guanine (CpG), is mediated by a member of the DNA methyltransferase (DNMT) family [38].…”