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


CONCLUSIONS
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.
Kwan
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Smooth Muscle Research Program, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. kwancy@fhc.mcmaster.ca
Kwan
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002733:Chlorophenols / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D011651:Pulmonary Artery
D002120:Calcium Channel Agonists; D002733:Chlorophenols; D007211:Indoles; C091710:4-chloro-3-ethylphenol; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid