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

J Lab Clin Med. 1988 Aug;112(2):157-67.

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 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antithrombins / pharmacology
  • Calcium / analysis*
  • Cell Adhesion
  • Cell Division
  • Cell Survival
  • Cells, Cultured
  • Cytopathogenic Effect, Viral
  • Endothelium, Vascular / analysis*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / microbiology
  • Histamine / pharmacology
  • Humans
  • Simplexvirus / growth & development
  • Spectrometry, Fluorescence
  • Temperature
  • Thrombin / antagonists & inhibitors
  • Thrombin / pharmacology
  • Umbilical Veins

Substances

  • Antithrombins
  • Histamine
  • Thrombin
  • Calcium