Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS)

- geNOps allow real-time imaging of eNOS-mediated NO
- formation in single cells.
- Impairment of mitochondrial Ca2+ uptake reduces NO
- synthesis by eNOS.
- Increased mitochondrial Ca2+ uptake facilitates Ca2+-triggered NO
- formation.
- Mitochondrial Ca2+ uptake does not affect eNOS phosphorylation.
- The link between mitochondria and eNOS activity remains unidentified.

Mitochondrial Ca2+ 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 Ca2+. The objective of this study was to verify whether or not mitochondrial Ca2+ uptake influences Ca2+-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 Ca2+ sensors to monitor single cell NO
- and Ca2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca2+-triggered NO
- increase independently of global cytosolic Ca2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca2+ sequestration and Ca2+-induced NO
- signals. The positive correlation between mitochondrial Ca2+ elevation and NO
- production was independent of eNOS phosphorylation at serine1177. Our findings emphasize that manipulating mitochondrial Ca2+ uptake may represent a novel strategy to control eNOS-mediated NO
- production.

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