title: 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.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Suphachai
authors.initials: S
authors.lastname: Charoensin
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Emrah
authors.initials: E
authors.lastname: Eroglu
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Marissa
authors.initials: M
authors.lastname: Opelt
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Bischof
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Corina T
authors.initials: CT
authors.lastname: Madreiter-Sokolowski
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Andrijana
authors.initials: A
authors.lastname: Kirsch
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Depaoli
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Saša
authors.initials: S
authors.lastname: Frank
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Astrid
authors.initials: A
authors.lastname: Schrammel
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Bernd
authors.initials: B
authors.lastname: Mayer
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Waldeck-Weiermair
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria. Electronic address: roland.malli@medunigraz.at.
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology; D057809:HEK293 Cells; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction
chemical_list: D015220:Calcium Channels; C034703:mitochondrial calcium uniporter; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D002118:Calcium
keywords: Calcium; ENOS; Endothelial nitric oxide production; GeNOps; Mitochondria