Key result
Knock-down of the mitochondrial calcium uniporter (MCU) and EMRE attenuated Ca2+-triggered NO• increase, whereas knock-down of MICU1 increased mitochondrial Ca2+ sequestration and NO• signals.
Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase, suggesting a potential strategy to control NO production.
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Mitochondrial Ca2+ uniport regulates endothelial NO in vitro; hypothesis-generating for MCU modulation, no clinical implications yet.
Mitochondrial Ca 2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO • ) production. However, it is not entirely clear if the organelles support or counteract NO • biosynthesis by taking up Ca 2+ . The objective of this study was to verify whether or not mitochondrial Ca 2+ uptake influences Ca 2+ -triggered NO • generation by endothelial NO • synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO • probes, the geNOps, and Ca 2+ sensors to monitor single cell NO • and Ca 2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP 3 )-generating agonist. Mitochondrial Ca 2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca 2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca 2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca 2+ -triggered NO • increase independently of global cytosolic Ca 2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca 2+ sequestration and Ca 2+ -induced NO • signals. The positive correlation between mitochondrial Ca 2+ elevation and NO • production was independent of eNOS phosphorylation at serine 1177 . Our findings emphasize that manipulating mitochondrial Ca 2+ uptake may represent a novel strategy to control eNOS-mediated NO • production.
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Charoensin et al. (2016) studied this question. siRNA-mediated knock-down of MCU, EMRE, and MICU1 was evaluated on Ca2+-triggered NO• generation. Knock-down of the mitochondrial calcium uniporter (MCU) and EMRE attenuated Ca2+-triggered NO• increase, whereas knock-down of MICU1 increased mitochondrial Ca2+ sequestration and NO• signals.
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