Acute tissue-type plasminogen activator release in human microvascular endothelial cells: the roles of Galphaq, PLC-beta, IP3 and 5,6-epoxyeicosatrienoic acid.

James A S Muldowney, Corrie A Painter, Elaine Sanders-Bush, Nancy J Brown, Douglas E Vaughan

Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA.

Thrombosis and haemostasis 2007 Feb

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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 Galphaq-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 Galphaq- 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 Galphaq inhibitory peptide, the PLC-beta antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Galphaq 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 microM 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 Galphaq-, PLC-beta-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Citation

James A S Muldowney, Corrie A Painter, Elaine Sanders-Bush, Nancy J Brown, Douglas E Vaughan. Acute tissue-type plasminogen activator release in human microvascular endothelial cells: the roles of Galphaq, PLC-beta, IP3 and 5,6-epoxyeicosatrienoic acid. Thrombosis and haemostasis. 2007 Feb;97(2):263-71