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Selected Publication:
Naghdi, S.
Ca2+ homeostasis in human vascular endothelial cells
[ Dissertation ] Medical University of Graz; 2010. pp. 132
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- Authors Med Uni Graz:
- Naghdi Shamim
- Advisor:
- Graier Wolfgang
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- Abstract:
- Ca2+ entry upon store depletion or SOCE (Store-operated Ca2+ entry) has a role in diverse range of cell function such as exocytosis, enzymatic activity, muscle contraction and apoptosis. On the other hand, mitochondrial Ca2+ handling has been proposed as an essential phenomenon in shaping temporal and spatial pattern of cytosolic Ca2+ in eukaryotic cells. Pharmacological tools, which disrupt mitochondrial Ca2+ signaling, have an inhibitory effect in the extent of SOCE. Molecular components of SOCE have been identified very recently. Upon ER Ca2+ depletion, the ER located Stromal interacting molecule 1 (STIM1) clusters and subplasmalemmal clusters interact with the plasma membrane Ca2+ channel Orai1 that , in turn, gets activated, resulting in Ca2+ entry. Nevertheless, mitochondrial Ca2+ sequestering effect on this specific pathway has not been investigated so far. The aim of this research was to study the effect of mitochondrial function (uptake/buffering) and motility on STIM1/Orai1- mediated SOCE. To achieve this aim, global and organelle Ca2+ signaling were recorded on high-resolution fluorescence microscopes using either fura-2/am or genetically encoded Ca2+ sensors that were targeted in the respective organelle. SOCE in cells expressing STIM1 and Orai1, exhibited less sensitivity to FCCP/oligomycine in comparison with control cells. In agreement, using different concentration of extracellular Ca2+ (0.5, 2, 10 and 20 mM) in nontransfected cells, revealed a reduced sensitivity of SOCE to mitochondrial poisoning at high concentration of extracellular Ca2+ concentration. It was found that application of the inhibitor of NCXmito, CGP 37157, blocked the FCCP-insensitive signal. Accordingly, upon large SOCE and under depolarizing condition (FCCP/oligomycin), mitochondrial Ca2+ accumulation was found to be sensitive to CGP 37157. In contrast, mitochondrial Ca2+ uptake was insensitive to inhibition of NCXmito under control condition. Immobilization of mitochondria using mAKAP-RFP-CAAX expression decreased mitochondrial Ca2+ uptake. However, it did not have any effect on SOCE-mediated Ca2+ entry. Depolarization of immobilized mitochondria in cells expressing mAKAP-RFP-CAAX, using FCCP/oligomycin had an inhibitory effect on SOCE mediated Ca2+ entry. This result indicate that mitochondrial Ca2+ is necessary for SOCE process in cells expressing STIM1 and Orai1, however, proximity, motility as well as the amount of local Ca2+ buffering of this organelle does not seem to play a role.