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 of 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.