Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells

Abstract

1. Intracellular Ca2+ ([Ca2+](i)) signals were studied with spatial resolution in bovine vascular endothelial cells using the fluorescent Ca2+ indicator fluo-3 and confocal laser scanning microscopy. Single cells were stimulated with the purinergic receptor agonist ATP resulting in an increase of [Ca2+](i) due to intracellular Ca2+ release front inositol 1,4,5-trisphosphate (IP3)-sensitive stores. ATP-induced Ca2+ release was quantal, i.e. submaximal 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 micrometres into non-stimulated regions. Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). CCE was initially colocalized with Ca2+ release. Following repetitive focal stimulation, however, CCE became detectable at remote sites where no Ca2+ release had been observed. In addition, the rate of Ca2+ store depletion with 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-dependent Ca2+ release and activation of CCE are controlled locally at the subcellular 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.