""""""""The role of calcium in the regulation of ion channels, downstream signaling, and gene expression was investigated. CAI, an inhibitor of nonvoltage-gated calcium influx, has been used as a tool to dissect calcium-mediated signaling. We have focused on the role of calcium on the regulation of endothelial cell function and have demonstrated that CAI inhibits endothelial cell proliferation, adhesion, motility, and collagenase expression. Intracellular free cytosolic calcium increases after attachment to type IV collagen, during the spreading process. This is inhibited by exposure to CAI initiated at completion of attachment. We have now demonstrated that calcium influx is necessary for formation of actin stress fibers during the spreading process. This is unique to type IV collagen, not occurring on type I collagen. Stress fiber formation can be expedited by agents that increase calcium influx and abrogated by CAI or other agents that chelate extracellular calcium or prevent influx. We have demonstrated that the biochemical effect occurs at the point of RhoA activation. We have linked our calcium-regulation of focal contact formation with the stress fiber observations by demonstration of a calcium influx sensitive partnership between FAK and p190RhoGAP. Further studies are ongoing to confirm these findings using molecular constructs to regulate the Rac-RhoA pathway in endothelial cells. We had hypothesized that extracellular Ca++ was regulatory to endothelial cells. We found that increasing extracellular Ca++ inhibited spreading and reduced motility but surprisingly found that extracellular Mg++ functioned as a chemoattractant for and to augment spreading of endothelial cells. A dose dependent effect of extracellular Mg++ was shown for chemotaxis, chemokinesis, and spreading on multiple substrata. Dissection of the pathways involved in Mg++-stimulated chemotaxis is in progress. Preliminary results indicate that inhibition of tyrosine phosphorylation with a tyrphostin abrogates migration as does treatment with pertussis toxin and a nonspecific inhibitor of protein kinase C isotypes. This suggests that Mg++ mediates its chemotactic function through a G-alpha-i linked receptor and a tyrosine kinase. Further studies are ongoing.""""""""
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