Measurement of intracellular calcium concentration in intact monolayers of human endothelial cells.
Intracellular calcium ([Ca++]i) plays an important role in signal transduction and cell activation. The measurement of [Ca++]i in intact monolayers of human umbilical vein endothelial cells with the fluorescent calcium-sensitive probe fura 2 has been evaluated. Monolayers provide a more physiologic cell preparation than suspensions and allow a greater variety of experimental manipulation. Basal [Ca++]i was 117 +/- 5 nmol/L, with a range from 40 to 280 nmol/L that was not affected by cell age (days of primary culture) or degree of confluence. Thrombin in concentrations of 0.005 to 5 NIH units/ml produced a dose-dependent increase in [Ca++]i up to a maximum of 1500 +/- 147 nmol/L; this increase was shown to depend in part on the concentration of extracellular calcium. The presence of antithrombin III at physiologic concentrations abolished responses to 0.5 NIH units/ml thrombin but had no effect on 5 NIH units/ml. The potential of this technique was demonstrated further by our ability to examine [Ca++]i responses in endothelial cells following infection with herpes simplex virus type 1, a virus implicated in vascular injury. After 18 hours' infection, the response to both thrombin and histamine was dramatically reduced despite a normal resting [Ca++]i. It is concluded that this method may be useful for detecting early and subtle changes in endothelial cell function under a variety of physiologic and pathologic conditions.
Wickham
N W
NW
Department of Medicine, University of Minnesota Medical School, Minneapolis.
Vercellotti
G M
GM
Moldow
C F
CF
Visser
M R
MR
Jacob
H S
HS
D000991:Antithrombins / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D002448:Cell Adhesion; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D003588:Cytopathogenic Effect, Viral; D004730:Endothelium, Vascular / Q000032:analysis* / Q000166:cytology / Q000382:microbiology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D018139:Simplexvirus / Q000254:growth & development; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014471:Umbilical Veins
D000991:Antithrombins; D006632:Histamine; D013917:Thrombin; D002118:Calcium