1. Intracellular Ca2+ ([Ca2+](i)) signals were studied with spatial resolut
ion in bovine vascular endothelial cells using the fluorescent Ca2+ indicat
or fluo-3 and confocal laser scanning microscopy. Single cells were stimula
ted with the purinergic receptor agonist ATP resulting in an increase of [C
a2+](i) due to intracellular Ca2+ release from inositol 1,4,5-trisphosphate
(IP3)-sensitive stores. ATP-induced Ca2+ release was quantal, i.e. submaxi
mal concentrations mobilized only a fraction of the intracellularly stored
Ca2+.
2. Focal receptor stimulation in Ca2+-free solution by pressure application
of-agonist-containing solution through a fine glass micropipette resulted
in a spatially restricted increase in [Ca2+](i). Ca2+ release was initiated
at the site of stimulation and frequently propagated some tens of micromet
res into non-stimulated regions.
3. Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). C
CE was initially colocalized with Ca2+ release. Following repetitive focal
stimulation, however, CCE became detectable at remote sites where no Ca2+ r
elease had been observed. In addition, the rate of Ca2+ store depletion wit
h repetitive local activation of release in Ca2+-free solution was markedly
slower than that elicited by ATP stimulation of the entire cell.
4. From these experiments it is concluded that both intracellular IP3-depen
dent Ca2+ release and activation of CCE are controlled locally at the subce
llular level. Moreover, redistribution of intracellular Ca2+ stored within
the endoplasmic reticulum efficiently counteracts local store depletion and
accounts for the spatial spread of CCE activation.