Measurement of intracellular calcium concentration in intact monolayers of human endothelial cells

N. W.R. Wickham, Gregory M Vercellotti, C. F. Moldow, M. R. Visser, Harry S Jacob

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)157-167
Number of pages11
JournalJournal of Laboratory and Clinical Medicine
Volume112
Issue number2
StatePublished - 1988

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