Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells.
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 from 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. 3. 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.
Hüser
J
J
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153,, USA. jhueser@luc.edu
Holda
J R
JR
Kockskamper
J
J
Blatter
L A
LA
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines; D002417:Cattle; D002478:Cells, Cultured; D004553:Electric Conductivity; D004721:Endoplasmic Reticulum / Q000502:physiology / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011651:Pulmonary Artery / Q000166:cytology; D058913:Purinergic Agonists
D002232:Carbocyanines; D005456:Fluorescent Dyes; D058913:Purinergic Agonists; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium