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

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