Endothelial Cell Responses to Inflammatory and Thrombotic Mediators — Evidence for Localisation of Cell-Cell Signals

  • Nicholas Wickham
Part of the NATO ASI Series book series (NSSA, volume 294)


Human umbilical vein endothelial cells (EC) were isolated and cultured on glass cover slips. This permitted insertion of EC into a cuvette in a thermostatically controlled cuvette holder which enabled calcium responses to be assessed in a SPEX scanning spectrofluorimeter using the calcium-fluorescent probe, Fura-2.1 In addition, EC phosphatidylinositol (PI) turnover and platelet activating factor (PAF) production were also measured using standard techniques based on incorporation of 3H-myo-inositol and 3H-acetate respectively. Thrombin stimulation of EC produced a dose-dependent rise in intracellular calcium between doses of 0.005 u/ml to 1 u/ml. Higher doses produced a more rapid and larger peak response and a correspondingly more rapid decline, reaching baseline much quicker than after lower doses, suggesting that following potentially damaging stimuli a scavenging mechanism may be being activated. A classical model of cellular activation involves a rise in intracellular calcium linked to an increase in PI turnover, however, these two signal transduction processes may be obligatory connected. Calcium transients may be detected in EC following stimulation with thrombin at doses as low as 0.005 u/ml, whereas an increase in PI turnover could not be detected until doses of thrombin of 0.1 u/m. or greater were used. Similarly, an increase in EC-PAF production was apparent which mirrored the dose-response to thrombin. When resting neutrophils (final concentration 1 × 107/ml) are suspended in the Hanks buffer surrounding the EC during an experiment, no calcium transient is detected. However, if the neutrophils are activated by the addition of the bacterial peptide, f-met—leu—phe (FMLP), then a low level transient calcium signal (25% above baseline) is generated.2


Human Umbilical Vein Endothelial Cell Platelet Activate Factor Signal Transduction Process Platelet Activate Factor Antagonist Endothelial Cell Response 
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Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Nicholas Wickham
    • 1
  1. 1.Department of Clinical OncologyThe Chinese University of Hong KongShatin, N.T.Hong Kong

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