Abstract 11427: Calcium Mobilization From Endoplasmic Reticulum Involves Calmodulin-mediated NADPH Oxidase-derived Reactive Oxygen Species Production in Porcine Aortic Endothelial Cells
Introduction: Previous studies indicate that calcium/calmodulin (Ca2+/CaM) mediates the phosphorylation and activation of NADPH oxidase (NOX). In endothelial cells, elevation of intracellular Ca2+ concentration ([Ca2+]i) level consists of two components, mobilization of Ca2+ from intracellular stores and subsequent store-operated Ca2+ entry (SOCE). However, little is known which component is required to regulate NOX-derived ROS production via Ca2+/CaM dependent pathway in endothelial cells.
Hypothesis: We hypothesized that Ca2+ mobilization from endoplasmic reticulum (ER), but not SOCE, is required to regulate NOX-derived ROS via Ca2+/CaM dependent pathway in porcine aortic endothelial cells (PAECs).
Methods: We evaluated the association between Ca2+/CaM mediated NOX-derived ROS production and Ca2+ mobilization from ER. We measured [Ca2+]i by fura-2/AM a production of ROS by C-DCDHF-DA and in primary cultured PAECs with a fluorescence imaging and analysis system.
Results: (1) In the presence of 1mM extracellular Ca2+ ([Ca2+]o), BK induced a rapid increase [Ca2+]i and followed by a sustained increase. However, in the absence of [Ca2+]o (0mM with EGTA 1mM), BK caused only a small and transient increase [Ca2+]i which was cause by Ca2+ mobilization from ER.
(2) BK (1μM) rapidly increased fluorescence intensity of C-DCDHF-DA compared with control. (150.2±51.3% and 107.2±5.6% of the baseline, respectively, p<0.05).
(3) BK-induced ROS production was inhibited by an inhibitor of NOX (VAS2870: 50μM) (125.5±9.9% of the baseline, respectively, p<0.05).
(4) When cells were exposed to BK with or without [Ca2+]o, there was no difference in BK-induced ROS production.
(5) In the absence of [Ca2+]o, BK-induced ROS production is inhibited by an inhibitor of calmodulin (W-7: 100μM) (121.3±13.1% of the baseline, p<0.05). Thapsigargin (an inhibitor of ER calcium ATPase: 1μM) and BAPTA/AM (100μM) eliminated BK-induced ROS production (110.0±5.1% and 115.7±9.5% of the baseline, respectively, p<0.05 vs 1mM [Ca2+]o).
Conclusions: The NOX-derived ROS production by BK is mediated via Ca2+/CaM dependent pathway. This was strictly regulated by Ca2+ mobilization from ER.
Author Disclosures: K. Odagiri: Speakers Bureau; Modest; Pfizer, GlaxoSmithKline. A. Hakamata: None. H. Watanabe: Research Grant; Significant; Ministry of Health, Labour and Welfare of Japan. Other Research Support; Modest; Teika Seiyaku, Sawai Seiyaku, Kaken Seiyaku, Nippon Shinyaku, Acterion, Daiichi Sankyo. Speakers Bureau; Modest; Pfizer, Acterion, Astellas Pharmaceuticals, GlaxoSmithKline, MSD.
- © 2015 by American Heart Association, Inc.