Intracellular Ca2+ distribution under pathological condition

Hertha Firnberg Position T 73 Intracellular Ca2+ distribution under pathological conditions Jolanta PALTAUF- DOBURZYNSKA 27.06.2000 The vascular endothelium is a very important regulator of metabolism between blood and muscle cells and decreases the resistance of the blood vessel. Endothelial cells release factors that regulate dilatation and contraction of the vessels and play a critical role in modulating both the inflammatory response and vasomator abnormalities that occur in coronary artery disease or risk factors for disease. This endothelial function is associated with changes of endathelial Ca2+ concentration. Numerous membrane proteins as well as intracellular enzymes are activated through Ca2+ released from intracellular stores. Endothelium is a direct target of oxygen free-radicals. Oxidation products may damage the ability of endothelial cells to produce or release nitric oxide and other relaxation / contraction factors. This damage of endothelium is a first step to atherosclerosis. A role for cellular oxidative stress mediated by glycoxydation or lipoxiclation has gained widespread acceptance. Up till now the cell response to high glucose or/and free cholesterol concentration has not been described in detail. Here a new aspect of endothelial Ca2+ signaling will be studied. The present project is designed to address questions about subplasmalemmal and cytosolic. Ca2+ distribution in endothelial cells loaded with high glucose or / and high cholesterol concentration. The changes of intracellular Ca2+ concentration will be visualized by the new method we will use. We will measure the changes in both intracellular Ca2+ - concentration and Ca2+ - distribution in single endothelial cells simultaneously with the fluorescence method using deconvolution microscopy. We will investigate the activation of subplasmalemmal and intracellular Ca2+ - dependent enzymes in correlation with endothelial dysfunction during hypercholesterolemia, diabetes mellitus or during both of these diseases (atherosclerosis in diabetes). This study may open a new understanding of activation of intracellular / subcellular compartments and their sensitivity during the above mentioned diseases. The investigations will be carried out in three steeps: 1) The effect of high glucose concentration on human endothelial cells by measuring transmembrane signaling, Ca2+ - oscillations, subplasmalemmal Ca2+ release from intracellular stores and Ca2+ distribution and localization of Golgi apparatus, mitachondria and endoplasmatic reticulum. 2) The effect of an enrichment of low density lipoprotein (LDL) or oxidized LDL (oxLDL) on membrane order. The experiments will include measurements of the subplasmalemmal and cytosolic Ca2+ distribution, Ca2+ oscillations, activation of superficial Ca2+ - stores (e.g. ryanodine stores) and subcellular gradients. 3) Influence of diabetes in atherosclerosis on human endothelial dysfunction, changes in intracellular Ca2+ signaling transduction, architecture of subplasmalemmal area, localization of intracellular compartments, activity of intracellular / subplasmalemmal stores ( Ryanodine- , IP3 -stores). In this Proiect soatially and temporally resolved measurements of local Ca2+ - concentration in freshly isolated endothelial / muscle cells from diabetic or hypercholesterolemic patients, will be used as a new method. Digital imaging, image deconvolution techniques and the 2D/3D reconstruction procedure will allow determination of intracellular / subcellular Ca2+ concentration and its distribution into cellular comoartments under normal and pathological conditions. We will show that not only the chances in the cell membrane but also the partial disruption of endoplasmatic reticulum and interrupted connection between superficial compartments and membrane channels are already the beginning of the cell damage. The spatial resolution of fluorescence imaoing is a great advantage when studvina the localization of ions and macromolecules and for imaging the three-dimensional orcanization of intracellular organelles.