Calcium Signaling in Endothelial Cells Involves Activation of Tyrosine Kinases and Leads to Activation of Mitogen-Activated Protein Kinases

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Abstract
The activation of endothelial cells following exposure to a variety of receptor-dependent and -independent stimuli is associated with the release of Ca 2+ from intracellular stores as well as the influx of Ca 2+ from the extracellular space. In the present study, we investigated the interaction between Ca 2+ signaling in cultured human umbilical vein endothelial cells and tyrosine phosphorylation. Stimulation of endothelial cells with either bradykinin (100 nmol/L), histamine (1 μmol/L), or the Ca 2+ -ATPase inhibitor thapsigargin (30 nmol/L) resulted in a slightly delayed but prolonged tyrosine phosphorylation of two low molecular weight proteins (≈42 and ≈44 kD). These proteins were identified by immunoprecipitation as the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase). The agonist-induced tyrosine phosphorylation of the 42-/44-kD doublet was sensitive to the tyrosine kinase inhibitors genistein (100 μmol/L) and piceatannol (10 μmol/L) and was inhibited by the removal of Ca 2+ from the extracellular medium. In fura 2–loaded endothelial cells, inhibition of tyrosine kinases attenuated Ca 2+ signaling after stimulation with either bradykinin (30 nmol/L) or thapsigargin (30 nmol/L). Since inhibition of tyrosine kinases specifically attenuates the plateau phase of the Ca 2+ response after stimulation, the effect of tyrosine kinase inhibition appeared to be mostly associated with the influx of Ca 2+ from the extracellular space. These data demonstrate that the signal transduction cascade initiated by receptor-dependent and -independent stimulation of endothelial cells includes the following: a tyrosine kinase inhibitor–sensitive transmembranous influx of Ca 2+ and the tyrosine phosphorylation of two cytosolic protein substrates identified as MAP kinases. Furthermore, on one hand, an increase in [Ca 2+ ] i was essential for tyrosine phosphorylation; on the other, the Ca 2+ influx was modulated by tyrosine phosphorylation. This finding documents the mutual dependence of these two crucial signaling pathways in endothelial cells.

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