The role of calcium in the regulation of ion channels, downstream signaling, and gene expression in endothelial cells has been under investigation. 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 regulated by changes in intracellular calcium due to calcium influx. This calcium influx is necessary for formation of actin stress fibers at the level of RhoA activation. This event 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 expand our dissection of the signaling events occurring during endothelial cell spreading on basement membrane collagen. 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. Migration to Mg++ is abrogated by inhibition of heterotrimeric signaling pathways by treatment with pertussis toxin, but not cholera toxin, suggesting that it is in the phospholipase pathway and not adenylyl cyclase regulation. Further, migration is blocked by d-erthyrosphingosine, an inhibitor of all three families of protein kinase C, and by tyrphostin B-48. Studies are in progress to distinguish between regulation of the Mg-ATPase and a potential Mg receptor. Studies of the role of other angiogeneis regulators on these signaling pathways has begun. - signal transduction, signaling, angiogenesis, calcium, motility,
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