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

Abstract

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.