Spatiotemporal Calcium Signaling in the Endothelium

Research project P 14586 Spariotemporal Calcium Signaling in the Endothelium Wolfgang GRAIER 09.10.2000 It is still fascinating that a molecule as simple as Ca2+ serves as a high selective intracellular mediator for a multitude of cellular functions. In the endothelium, the main non-nerval regulator of vascular homeostasis, the varity of Ca2+ regulatory function is convincingly demonstrated by its property to initiate the production of vasoactive factors such as the relaxing nitric oxide as well as the contracting endothelin. Hence, Ca2+ triggers, among others, the activation of transcription factors, the activity of mitogenic enzymes, the binding of transcription factors and counteracts apoptosis. While such multiple, sometimes oppositional actions of the Ca2+ signal emphasize Ca2+-sensitive mechanisms in a very sophisticated manner. This "Ca2+ paradox" for multiple but still highly specific actions of intracellular Ca2+ is proposed to be achieved by a localized Ca2+ signaling. In respect to its central role and outstanding importance, it is not surprising that endothelial cells express several isoforms of at least 8 Ca2+-shuttling proteins, several Ca2+-sensitive ion channels and four Ca2+-containg organelles that concertedly control localized/global Ca2+. This project is designed to elucidate the functional coupling of the contributors to endothelial Ca2+ signaling and to explore the role of localized/global Ca2+ signaling for acute and long lasting (e.g. gene expression) cell function. In order to reach the project`s aim, an instrumental device that allows fast high resolution measurement of up to 4 parameters, the organelle-specific expression of Ca2+-shuttling proteins (virus-mediated gene transfer) will be developed. Hence, a "tissue culture model" will be elaborated that allows the in situ gene transfection in a function and gene expression, to be compared with the data obtained in single cell culture. In addition the spatial Ca2+ signaling as an initial target of changes in endothelial function in diabetes mellitus will be studied. To know how endothelial cell handle spatial Ca2+ will increase our understanding of the selective cell activation taken place in vivo and may open a new principle of a mechanism of vascular complications in life-threatening diseases, such as diabetes.

In view of the intriguing specificity of Ca2+ as messenger and its versatile and ubiquitous contribution to most signaling pathways/cell functions, the local activation of Ca2+ sensitive proteins/pathways by spatial Ca2+ fluctuations was studied in many cell types. Aim of the present project was to investigate the appearance of such locally limited Ca2+ signaling as key mechanism of cell regulation in endothelial cells and to explore the mechanisms regulating such "hot/low Ca2+ spots". Following the concept that the multi-functionality of Ca2+ in cell physiology is achieved, at least in part, by the generation of spatial Ca2+ gradients that are isolated from the usually called "cytosolic" Ca2+, we have utilized and improved organelle targeted Ca2+-sensing proteins and established their measurement on a high resolution and fast laser scanning device, which was further equipped with a electrophysiological setup to monitor ion movements across the cell membrane on distinct and clear defined locations at the cell surface. As all these techniques were designed to conduct experiments in single living cells, various techniques that allow monitoring of Ca2+-sensitive signaling pathways (e.g. tyrosine kinases, transcription factors, mitochondrial function or protein folding) at the cellular and subcellular level have been established and/or developed in course of this project. Our results indicate that in endothelial cells intracellular Ca2+ homeostasis is strictly controlled by a concerted action between the endoplasmic reticulum, mitochondria and plasmalemmal cation channels. In particular, upon cell stimulation, the endoplasmic reticulum is capable to create a high Ca2+ microenvironment beneath the cell membrane that yields activation of Ca2+-activated K+ channels, which, in turn, results in cell hyperpolarization, the driving force for Ca2+ entry through the non-voltage-gated entry pathway. Notably, Ca2+ influx through these Ca2+-inhibited Ca2+ channel(s) was found to be essentially maintained to a Ca2+-buffering function of subplasmalemmal domains of mitochondria. Mitochondrial Ca2+ uptake initiates ATP production and is of transient nature as Ca2+ is subsequently released vectorially towards neighboring ER domains that facilitate mitochondrial Ca2+ extrusion by a gaps of low Ca2+ established by SERCA between the organelles. As a consequence of such subcellular, spatial and tightly controlled Ca2+ signaling, various Ca2+-dependent cell functions were found to be distinctly regulated and, thus, the high complexicity of subcellular Ca2+ regulation was found to be a key for divers cell functions as well as a target for cell dysfunction under various circumstances.