Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.

Abstract

Abstract. Thrombin stimulates cultured endothelial ceils (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester-but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8/zg/ml for 5-15 min at 37~ which allow the introduction of small extracellular molecules without the loss of large intraceUular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.

Small GTP-Binding Protein Ral Modulates Regulated Exocytosis of von Willebrand Factor by Endothelial Cells

“…12,15 Recently, a binding site for calmodulin on Ral has been detected, and calmodulin has been shown to enhance the binding of GTP to Ral. 23,24 We investigated whether thrombin-induced activation of Ral is affected by antagonists of calmodulin.…”

cAMP-induced secretion of endothelial von Willebrand factor is regulated by a phosphorylation/dephosphorylation switch in annexin A2

“…2,3 Exocytosis of WPBs is stimulated by a varied set of agonists that can be separated into 2 main groups: those acting by elevating the free concentration of cytosolic Ca 21 and those acting by raising cyclic adenosine monophosphate (cAMP) levels. 4,5 For instance, histamine and thrombin have been shown to mediate secretion of WPBs by increasing the levels of cytosolic free Ca 21 , 6,7 whereas release of WPBs induced by secretagogues such as epinephrine or forskolin involves cAMP-dependent signaling. 8,9 Significant differences exist between Ca 21 -and cAMP-evoked WPB exocytosis and, whereas several cellular factors participating in the Ca 21 -regulated secretion of WPBs have been identified, much less is known about the molecular mechanisms underlying the cAMP-dependent exocytosis of WPBs.…”

Regulated von Willebrand Factor Secretion Is Associated With Agonist-Specific Patterns of Cytoskeletal Remodeling in Cultured Endothelial Cells

“…A role for calmodulin in mediating the Ca 2ϩ effect is suggested by the inhibitory action of a calmodulin-binding peptide on vWF release in permeabilized ECs. 2 The molecular mechanisms distal to the activation of calmodulin have so far not been studied.…”

“…One class of agonists, identified in in vitro studies, includes thrombin, histamine, and other mediators of inflammation and/or thrombosis. [2][3][4] Their effect seems to be mediated by a rise in cytosolic free Ca 2ϩ ([Ca 2ϩ ] i ). 2,5 A rise in [Ca 2ϩ ] i induced by Ca 2ϩ ionophores, such as A23187 or ionomycin, is sufficient to induce acute vWF release.…”

“…[2][3][4] Their effect seems to be mediated by a rise in cytosolic free Ca 2ϩ ([Ca 2ϩ ] i ). 2,5 A rise in [Ca 2ϩ ] i induced by Ca 2ϩ ionophores, such as A23187 or ionomycin, is sufficient to induce acute vWF release. 6,7 Thrombin-induced vWF release is associated with a rise in [Ca 2ϩ ] i and can be prevented by preincubation with intracellular Ca 2ϩ chelators.…”