INTERDEPENDENCE OF CALCIUM SIGNALING AND PROTEIN-TYROSINE PHOSPHORYLATION IN HUMAN ENDOTHELIAL-CELLS

The signal transduction cascade which initiates trans membraneous infl ux of Ca2+ into endothelial cells in response to the discharge of intr acellular Ca2+ stores is thought to involve a step sensitive to tyrosi ne kinase inhibition. We investigated the interrelationship between Ca 2+ signaling and protein tyrosine phosphorylation following cell stimu lation with either the receptor-dependent agonist, bradykinin, or the protein-tyrosine phosphatase inhibitor, phenylarsine oxide. In culture d human endothelial cells phenylarsine oxide instigated a concentratio n-dependent increase in the intracellular concentration of free Ca2+ ( [Ca2+](i)). This increase in [Ca2+](i) was not associated with the tyr osine phosphorylation of phospholipase C gamma, enhanced formation of inositol 1,4,5-trisphosphate, or the rapid depletion of intracellularl y stored Ca2+ but was coincident with the enhanced and prolonged tyros ine phosphorylation of a number of cytoskeletal proteins. In bradykini n-stimulated cells the tyrosine phosphorylation of the same cytoskelet al proteins (most notably 85- and 100-kDa proteins) was transient when cells were stimulated in the presence of extracellular Ca2+, was main tained under Ca2+-free conditions, and was reversed following readditi on of extracellular Ca2+. These data suggest that the tyrosine phospho rylation of 2 cytoskeletal proteins is determined by the level of Ca2 present in intracellular stores thus indicating a critical role for t yrosine phosphorylation in the control of capacitative Ca2+ entry in e ndothelial cells.