Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Charoensin, S.
Single-Cell Investigation of Endothelial Nitric Oxide Dynamics
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Graz Medical University; 2018. pp.
[OPEN ACCESS]
FullText
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Graier Wolfgang
- Malli Roland
- Altmetrics:
- Abstract:
- Mitochondrial calcium (Ca2+) uptake is a sophisticated mechanism that shapes both local and global Ca2+ signals, which control a variety of signaling pathways. Nevertheless, in the intact endothelial cell, it is not clear if mitochondria by taking up Ca2+ promote or counteract nitric oxide (NO•) synthesis, which is an essential process in vascular homeostasis. Therefore, the present study aimed to investigate the role of mitochondrial Ca2+ uptake on Ca2+-triggered NO• production in an immortalized endothelial cell line (EA.hy926) and respective primary human umbilical vein endothelial cells (HUVECs). For this purpose, single live-cell imaging for both NO• and Ca2+ was performed using recently developed genetically-encoded fluorescent NO• probes (geNOps) and a mitochondrial Ca2+ sensor (4mtD3cpv), respectively. Mitochondrial Ca2+ uptake is exquisitely controlled by several proteins of the inner mitochondrial membrane. So, in this study, mitochondrial Ca2+ uptake was manipulated using siRNAs against proteins that form the mitochondrial Ca2+ uniporter (MCU) complex and its associated regulatory proteins. Silencing of MCU and essential MCU regulator (EMRE) resulted in a significant decrease of mitochondrial Ca2+ signals and cytosolic NO• formation in response to adenosine triphosphate (ATP)-triggered Ca2+ mobilization in endothelial cells. This finding was confirmed by using antimycin and oligomycin, which collapse the mitochondrial membrane potential, thereby blocking Ca2+ uptake by mitochondria. Down-regulation of mitochondrial Ca2+ uptake 1 (MICU1), a negative regulator of the MCU complex, enhanced mitochondrial Ca2+ uptake and Ca2+-evoked NO• formation in both cell models. In conclusion, these results indicate that the endothelial NO• generation is indeed dependent on mitochondrial Ca2+ uptake. More importantly, the present study signifies a role of the MCU complex on endothelial NO• synthase (eNOS)-mediated NO• formation that might be a target for vascular function improvement.