Acute tissue-type plasminogen activator release in human microvascular endothelial cells: the roles of Galphaq, PLC-beta, IP3 and 5,6-epoxyeicosatrienoic acid.
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.
Muldowney
James A S
JA
Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA.
Painter
Corrie A
CA
Sanders-Bush
Elaine
E
Brown
Nancy J
NJ
Vaughan
Douglas E
DE
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001685:Biological Factors / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D008833:Microcirculation / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D054799:Phospholipase C beta; D011188:Potassium / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D001685:Biological Factors; D007527:Isoenzymes; C067410:endothelium-dependent hyperpolarization factor; D009569:Nitric Oxide; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; D015544:Inositol 1,4,5-Trisphosphate; D011464:Epoprostenol; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015126:8,11,14-Eicosatrienoic Acid; D011188:Potassium