Abstract
Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca(2+) or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.
Highlights
Ca2ϩ- and cAMP-raising agonists promote exocytosis of Weibel-Palade bodies from endothelial cells
Previous work from our group has indicated that Weibel-Palade bodies (WPBs) exocytosis in response to cAMP-mediated agonists is partly controlled by a protein kinase A (PKA)-dependent signaling pathway that eventually leads to the activation of RalA, a small GTPase that co-sediments with WPBs in density gradients (10 –12)
exchange protein activated by cAMP (Epac) and von Willebrand factor (VWF) Secretion—Exocytosis of WPBs occurs following triggering of G protein-coupled proteins of the Gs subtype, which elevate intracellular cAMP levels and promote PKA-dependent activation of RalA [4, 5, 12]
Summary
Ca2ϩ- and cAMP-raising agonists promote exocytosis of Weibel-Palade bodies from endothelial cells. Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca2؉ or cAMP. We have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac and its substrate, the small GTPase Rap. Down-regulation of Rap expression and prevention of Rap activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP. Previous work from our group has indicated that WPB exocytosis in response to cAMP-mediated agonists is partly controlled by a protein kinase A (PKA)-dependent signaling pathway that eventually leads to the activation of RalA, a small GTPase that co-sediments with WPBs in density gradients (10 –12). We explored a potential role for the cAMP-guanine nucleotide exchange factor Epac and its substrate Rap in the regulation of WPB exocytosis by human primary endothelial cells
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