Acute tissue-type plasminogen activator release in human microvascular endothelial cells: The roles of Gαq, PLC-β, IP3 and 5,6-epoxyeicosatrienoic acid

James A S Muldowney, Corrie A. Painter, Elaine Sanders-Busch, Nancy J. Brown, Dauglas E. Vaughan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

The acute physiologic release of tissue-type plasminogen activator (t-PA) from the endothelium is critical for vascular homeostasis. This process is prostacyclin- and nitric oxide (NO)-independent in humans. It has been suggested that calcium signaling and endothelial-derived hyperpolarizing factors (EDHF) may play a role in t-PA release. G-protein-coupled receptor-dependent calcium signaling is typically Gαq-dependent. EDHFs have been functionally defined and in various tissues are believed to be various regioisomers of the epoxyeicosatrienoic acids (EETs).We tested the hypothesis in vitro that thrombin-stimulated t-PA release from human microvascular endothelial cells (HMECs) is both Gαq- and EDHF-dependent. Conditioned media was harvested following thrombin stimulation, and t-PA antigen was measured by ELISA.Thrombin-induced t-PA release was limited by a membrane-permeable Gαq inhibitory peptide, the PLC-β antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Gαq agonist Pasteurella toxin modestly induced t-PA release. The cytochrome P450 (CYP450) inhibitor, miconazole, and the arachidonic acid epoxygenase inhibitor MS-PPOH inhibited thrombin-stimulated t-PA release, while 5,6-EET-methyl ester stimulated t-PA release. The 5,6- and 14,15-EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid, inhibited t-PA release at the 100 μM concentration. However, thrombin-stimulated t-PA release was unaffected by the prostacyclin and NO inhibitors ASA and L-NAME, as well as the potassium channel inhibitors TEA, apamin and charybdotoxin. These studies suggest that thrombin-stimulated t-PA release is Gα q-, PLC-β-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Original languageEnglish (US)
Pages (from-to)263-271
Number of pages9
JournalThrombosis and Haemostasis
Volume97
Issue number2
DOIs
StatePublished - Feb 2007

Keywords

  • Epoxyeicosa trienoic acids (EET's)
  • Pharmacology
  • Protease-activated receptor
  • Signal transduction
  • Thrombin
  • Tissue-type plasminogen activator

ASJC Scopus subject areas

  • Hematology

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