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

Aim: To study the spatial and temporal distribution of intracellular Ca2+ concentration 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 Ca2+ 1-2 mmol.L-1 to BPAE cells, which were exposed to Ca(2+)-free medium containing egtazic acid, resulted in a transient elevation of cytosolic Ca2+ concentration, which rapidly returned to the resting level. Biphasic elevation (a larger transient phase followed by a smaller sustained phase) of intracellular Ca2+ concentration was observed upon the addition of ATP (via activation of surface membrane receptor). 4-Chloro-3-ethyl phenol (CEP; an activator of Ca(2+)-induced Ca2+ channels) potently induced elevation of Ca2+ level. Cyclopiazonic acid (CPA; an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump) offered a more sustained elevation of Ca2+. In most cases, the highest level of Ca2+ elevation was observed around the cell peripheries, sometimes at rest and particularly upon stimulation. Ca2+ elevation associated with nuclear complex seemed to be higher compared to that in the cytosolic compartment.

Conclusion: Changes of cell Ca2+ upon stimulation by various agents that acted at different intracellular sites were found to be temporarily and spatially heterogenous among BPAE cells. At the single cell level, Ca2+ elevation seemed to occur initially near the peripheral region followed by the nuclear region. This study raised the possibility that nuclear Ca2+ and cytosolic Ca2+ might be regulated independently in BPAE cells.