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

Publication ,  Journal Article
Birch, KA; Pober, JS; Zavoico, GB; Means, AR; Ewenstein, BM
Published in: J Cell Biol
September 1992

Thrombin stimulates cultured endothelial cells (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 micrograms/ml for 5-15 min at 37 degrees C) which allow the introduction of small extracellular molecules without the loss of large intracellular 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.

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Published In

J Cell Biol

DOI

ISSN

0021-9525

Publication Date

September 1992

Volume

118

Issue

6

Start / End Page

1501 / 1510

Location

United States

Related Subject Headings

  • von Willebrand Factor
  • Umbilical Veins
  • Thrombin
  • Sulfonamides
  • Signal Transduction
  • Protein Kinase C
  • Peptide Fragments
  • Naphthalenes
  • Myosin-Light-Chain Kinase
  • Molecular Sequence Data
 

Citation

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Birch, K. A., Pober, J. S., Zavoico, G. B., Means, A. R., & Ewenstein, B. M. (1992). Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. J Cell Biol, 118(6), 1501–1510. https://doi.org/10.1083/jcb.118.6.1501
Birch, K. A., J. S. Pober, G. B. Zavoico, A. R. Means, and B. M. Ewenstein. “Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.J Cell Biol 118, no. 6 (September 1992): 1501–10. https://doi.org/10.1083/jcb.118.6.1501.
Birch, K. A., et al. “Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.J Cell Biol, vol. 118, no. 6, Sept. 1992, pp. 1501–10. Pubmed, doi:10.1083/jcb.118.6.1501.
Birch KA, Pober JS, Zavoico GB, Means AR, Ewenstein BM. Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. J Cell Biol. 1992 Sep;118(6):1501–1510.

Published In

J Cell Biol

DOI

ISSN

0021-9525

Publication Date

September 1992

Volume

118

Issue

6

Start / End Page

1501 / 1510

Location

United States

Related Subject Headings

  • von Willebrand Factor
  • Umbilical Veins
  • Thrombin
  • Sulfonamides
  • Signal Transduction
  • Protein Kinase C
  • Peptide Fragments
  • Naphthalenes
  • Myosin-Light-Chain Kinase
  • Molecular Sequence Data