Introduction:
Previous studies indicate that calcium/calmodulin (Ca
2+
/CaM) mediates the phosphorylation and activation of NADPH oxidase (NOX). In endothelial cells, elevation of intracellular Ca
2+
concentration ([Ca
2+
]
i
) level consists of two components, mobilization of Ca
2+
from intracellular stores and subsequent store-operated Ca
2+
entry (SOCE). However, little is known which component is required to regulate NOX-derived ROS production via Ca
2+
/CaM dependent pathway in endothelial cells.
Hypothesis:
We hypothesized that Ca
2+
mobilization from endoplasmic reticulum (ER), but not SOCE, is required to regulate NOX-derived ROS via Ca
2+
/CaM dependent pathway in porcine aortic endothelial cells (PAECs).
Methods:
We evaluated the association between Ca
2+
/CaM mediated NOX-derived ROS production and Ca
2+
mobilization from ER. We measured [Ca
2+
]
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 Ca
2+
([Ca
2+
]
o
), BK induced a rapid increase [Ca
2+
]
i
and followed by a sustained increase. However, in the absence of [Ca
2+
]
o
(0mM with EGTA 1mM), BK caused only a small and transient increase [Ca
2+
]
i
which was cause by Ca
2+
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 [Ca
2+
]
o
, there was no difference in BK-induced ROS production.
(5) In the absence of [Ca
2+
]
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 [Ca
2+
]
o
).
Conclusions:
The NOX-derived ROS production by BK is mediated via Ca
2+
/CaM dependent pathway. This was strictly regulated by Ca
2+
mobilization from ER.
Genistein Inhibits Expressions of NADPH Oxidase p22phox and Angiotensin II Type 1 Receptor in Aortic Endothelial Cells from Stroke-Prone Spontaneously Hypertensive Rats
