Dynamic digital fluorescence ratio imaging of cell calcium in vascular endothelial cells

AIM: To study the spatial and temporal distribution of intracellular Ca2+ c oncentration in cultured bovine pulmonary artery endothelial ( BPAE) cells. METHODS: Cultured BPAE cells were loaded with Fura-2 and observed under an inverted microscope coupled to a microfluorimeter, which enables pixel-to- pixel ratio imaging of the BPAE cells in real time. RESULTS: Addition of Ca 2+ 1 - 2 mmol.L-1 to BPAE cells, which were exposed to Ca2+-free medium con taining egtazic acid, resulted in a transient elevation of cytosolic Ca2+ c oncentration, which rapidly returned to the resting level. Biphasic elevati on (a larger transient phase followed by a smaller sustained phase) of intr acellular Ca2+ concentration was observed upon the addition of ATP (via act ivation of surface membrane receptor). 4-Chloro-3-ethyl phenol (CEP; an act ivator of Ca2+-induced Ca2+ channels) potently induced elevation of Ca2+ le vel. Cyclopiazonic acid (CPA; an inhibitor of endoplasmic reticulum Ca2+-AT Pase pump) offered a more sustained elevation of Ca2+. In most cases, the h ighest level of Ca2+ elevation was observed around the cell peripheries, so metimes at rest and particularly upon stimulation. Ca2+ elevation associate d with nuclear complex seemed to be higher compared to that in the cytosoli c compartment. CONCLUSION: Changes of cell Ca2+ upon stimulation by various agents that acted at different intracellular sites were found to be tempor arily and spatially heterogeneous among BPAE cells. At the single cell leve l, Ca2+ elevation seemed to occur initially near the peripheral region foll owed by the nuclear region. This study raised the possibility that nuclear Ca2+ and cytosolic Ca2+ might be regulated independently in BPAE cells.