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General Research Interests

Our general interest is the regulation and function of Ca2+ as one of the nature's most versatile messenger. Right from the beginning of our scientific activities, we got fascinated how a simple messenger, as simply as Ca2+, can serve as such a specific and manifold messenger.

Calcium research is challenging by various means:

1st, to measure Ca2+ signaling in living cells, organelles or even smaller spaces requires continuous improvements of techniques used,

2nd
to understand the molecular principles of Ca2+ regulation, one needs to identify/characterize the proteins that are involved, and

3rd
, to understand how Ca2+ finally affects signal transduction one needs to explore Ca2+-sensitive proteins that translate a Ca2+ signal to protein activity.

General Background on the role of spatial and temoral aspects of the Ca2+ signaling

Due to its fascinating versatility Ca2+ embodies an enfant terrible among the cellular ions. As in virtually every cell type, Ca2+ operates as a crucial messenger for numerous pivotal functions in endothelial cells. There Ca2+ regulates the production of vasoactive compounds (1), activation of transcription factors (e.g. NFkB; 2; CREB; 3-5), major signal transduction pathways (e.g. MAPK; 6, 7), protein folding (8-11), apotosis (12) and the function of ion channels (13). Such exciting variability of Ca2+ as a messenger is even more impressive if one considers the precision by which Ca2+-sensitive mechanisms are regulated. But how can a molecule as simple as Ca2+ be the mediator of such a complex and precise machinery?

In pancreatic acinar cells (14), cardiac myocytes (15), smooth muscle (16) and HeLa cells (17) Ca2+ signaling was found to constitute a multitude of local, highly controlled processes that include ion channels, pumps and organelles. In agreement with these landmark contributions, the complexity of endothelial Ca2+ homeostasis was investigated (18, 19). As in most other non-excitable cells, the predominant sources for elevation of [Ca2+]cyto in endothelial cells are the ER and the extracellular space (1). Besides the ubiquitous pathway of IP3-triggered emptying of the ER and subsequent activation of capacitative Ca2+ entry (CCE; 20) that achieves long lasting elevation of [Ca2+]cyto, we described the appearance of local Ca2+ gradients in endothelial cells (21-24). Moreover, we found preliminary evidence of an inter-organelle Ca2+ crosstalk between the ER and the mitochondria that represents a key phenomenon in cellular Ca2+ homeostasis. Such inter-organelle Ca2+ communication has been also described in HEK293 and HeLa cells (25-28) and emphasizes that in non-excitable cells these different cellular Ca2+ handling organelles might cooperatively control cellular Ca2+ homeostasis to accomplish specific Ca2+-regulation of multiple cell functions.

We investigated the diverse functions of subplasmalemmal domains of the ER and the mitochondria. We and other described that superficial domains of the ER (i.e. subplasmalemmal Ca2+ control unit, SCCU; 21-24, 29-31) and mitochondria (i.e. mitochondrial Ca2+ buffering unit, MCBU; 24) accomplish the initiation and maintenance of opposite subplasmalemmal Ca2+ gradients. Moreover, the orchestration of such opposite Ca2+ gradients obviously need a specific interplay within these organelles, particular architectural organization and controlled movements.

Intracellular Ca2+ cycling
Intracellular Ca2+ signaling is much more than the release of Ca2+ from intracellular Ca2+ stores. In most cells, agonist-induced formation of inositol 1,4,5-trisphospate triggers intracellular Ca2+ mobilization from the ER. Subsequently to the emptying of the ER, a signal is generated that initiates the so called capacitative Ca2+ entry (Puney JW Jr.). Although the exact molecular nature of these channel(s) is still unclear, there are exciting new data recently published (e.g. Peinelt et al, Mercer et al, Soboloff et al.) that make us believe that the puzzle of the nature of these channels is soon solved. Based on data that suggested an involvement of mitochondria on the maintenance of Ca2+-inhibitable capacitative Ca2+ entry (Hoth et al, Gilabert et al) we have described that subplasmalemmal domains of the mitochondria indeed sequester entering Ca2+ very efficiently (Malli et al). Moreover, mitochondria deliver Ca2+ vectorially towards the ER to achieve rapid Ca2+ store refilling (Malli et al). Notably, under conditions of reduced Ca2+ entry, ER refilling is still accomplished at the cost of cytosolic Ca2+ elevation (Malli & Frieden et al).
On the other hand, subplasmalemmal Ca2+ release by superficial ER is essential to sptially activate Ca2+-activated K+ channels (Frieden et al) and, consequently accomplish membrane hyperpolarization, which increases the driving force for Ca2+ to enter the cell through capacitative Ca2+ entry channels.
Obviously, mitochondria are active contributors of intracellular Ca2+ signaling and their contribution, by far, exceeds that of an passive Ca2+ sink, as which these organelles were thought a long time. Consequently, one should take a closer look on the Ca2+ signal these organelle:

Mitochondrial Ca2+ signaling
Mitochondrial Ca2+ homeostasis is irresolvable associated with two great Italian scientists, Profs. Tullio Pozzan and Rosario Rizzuto. With the landmark introduction of aequorin as targeted Ca2+ sensor and the subsequent outstanding work of Profs. Roger Y. Tsien and Atsushi Miyawaki measurements of mitochondrial Ca2+ became available for many scientists. Nowadays. these techniques are pretty frequent and helped to reveal the role of mitochondrial in intracellular Ca2+ signaling. In endothelial cells, the intracellular Ca2+ cycling (see above) is driven by the property of mitochondria to sequester and to release Ca2+. However, despite the striking functional proofs for the existence of mitochondrial Ca2+ carrier (e.g. Kirichok et al), the actual molecular nature of these proteins is unknown. Surprisingly, there are already quite selective pharmacological tools that inhibit or activate mitochondrial Ca2+ shuttling proteins (CGP37157, SB202190). Nevertheless, the identification of the molecular nature of mitochondrial ion carriers is one of the greatest task in the field of Ca2+ signaling and we accept this challenge...

Pathological aspects of Ca2+ signaling in endothelial cells
Global endothelial Ca2+ signaling has been found to represent one of the initial targets in the genesis of vascular complications in e.g. diabetes mellitus (32-46), septic shock (47) and atherogenesis (48). Despite such overwhelming evidence that the distorted Ca2+ homeostasis accounts, at least in part, for endothelial dysfunction in the respective diseases, a detailed analysis regarding the molecular and local aspects of the alterations in Ca2+ signaling and their consequences for Ca2+-triggered cell function needs further attention. The consequences of alterations in spatial or organelle Ca2+ signaling are not always easy to estimate. In endothelial and smooth muscle cells isolated from human arteries alterations in subplasmalemmal Ca2+ signaling were reported in diabetes mellitus (Fleichhacker et al) and hypercholesterolemia (Fleichhacker et al). Interestingly most changes were associated with the occurrence of elevated superoxide anion production that distinctly alters endothelial, smooth muscle and platelet Ca2+ homeostasis (Wascher et al, Graier et al, Schaeffer et al, Graier et al) and represent an intercellular messenger within the vessel wall (Schaeffer et al).

The role of mitochondria in pathology
In line with the changes/dysfunction of endothelial cells upon exposure to elevated D-glucose, impressive changes in mitochondrial structure, kinetics, Ca2+ signaling and ROS (reactive oxygen species) production occure (Paltauf-Doburzynska et al). However, it is not entirely clear whether or not the reported changes in mitochondrial functions and structure are subsequent to an cellular dysfunction or represent the initial step on the series of events that finally end with cell damage. In other words, the role of mitochondria in pathology seems to be a thrilling one while we still do not know exactly whether or not mitochondria represent initiators or target of cellular dysfunction under pathological conditions. For example, an inhibition of mitochondrial Ca2+ transit diminished capacitative Ca2+ entry (Malli et al), ER Ca2+ refilling (Malli et al) and protein folding (Osibow et al). Considering that the role of mitochondria in cellular metabolism, we expect these organelle to represent the interface between metabolism and (Ca2+-dependent) signal transduction. We have just submitted a grant on this topics....we´ll see.

References
Note: we apologize for this very subjective and artificial selection that does not claim to be entirely correct in terms of citation the first reports or best/greatest contribution to the field
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21.       Frieden, M. & Graier, W. F. Subplasmalemmal ryanodine-sensitive Ca2+ release contributes to KCa channel activation in a human umbilical vein endothelial cell line. J. Physiol. 524, 715-724 (2000).
22.       Frieden, M., Malli, R., Samardzija, M., Demaurex, N. & Graier, W. F. Subplasmalemmal endoplasmic reticulum controls KCa channel activity upon stimulation with moderate histamine concentration in a human umbilical vein endothelial cell line. J. Physiol. 540, 73-84 (2002).
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30.       Paltauf-Doburzynska, J., Posch, K., Paltauf, G. & Graier, W. F. Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells. J. Physiol. Lond. 513, 369-379 (1998).
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