Warning: Constant WP_DEBUG already defined in /customers/b/1/8/ngfi.eu/httpd.www/wp-config.php on line 87 Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS) - Next Generation Fluorescence Imaging

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 NOgeneration 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 NOincrease 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. Read more…