Epoxyeicosatrienoic acids regulate Trp channel dependent Ca2+ signaling and hyperpolarization in endothelial cells.
OBJECTIVE
An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells.


RESULTS
In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca(2+) influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition. Similar effects were observed in cultured endothelial cells overexpressing CYP2C9, but not in CYP2C9-deficient cells, and were prevented by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid as well as by the cAMP antagonist, Rp-cAMPS. The effects on Ca(2+) were mirrored by prolongation of the bradykinin-induced hyperpolarization. Ruthenium red and the combination of charybdotoxin and apamin prevented the latter effect, suggesting that Trp channel activation increases Ca(2+) influx and prolongs the activation of Ca(2+)-dependent K(+) (K(Ca)) channels. Indeed, overexpression of CYP2C9 enhanced the agonist-induced translocation of a TrpC6-V5 fusion protein to caveolin-1-rich areas of the endothelial cell membrane, which was prevented by Rp-cAMPS and mimicked by 11,12-EET.


CONCLUSIONS
Elevated EET levels regulate Ca(2+) influx into endothelial cells and the subsequent activation of K(Ca) channels, via a cAMP/PKA-dependent mechanism that involves the intracellular translocation of Trp channels.
Fleming
Ingrid
I
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. fleming@em.uni-frankfurt.de
Rueben
Alexandra
A
Popp
Rüdiger
R
Fisslthaler
Beate
B
Schrodt
Susanne
S
Sander
Anna
A
Haendeler
Judith
J
Falck
John R
JR
Morisseau
Christophe
C
Hammock
Bruce D
BD
Busse
Rudi
R
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000218:Adamantane / Q000031:analogs & derivatives / Q000494:pharmacology; D001030:Apamin / Q000494:pharmacology; D001685:Biological Factors / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D051242:Caveolin 1 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004537:Eicosanoic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials; D049990:Membrane Transport Modulators / Q000494:pharmacology; D008825:Miconazole / Q000494:pharmacology; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014665:Vasodilator Agents / Q000378:metabolism*
D001685:Biological Factors; D051242:Caveolin 1; D065607:Cytochrome P-450 Enzyme Inhibitors; D004537:Eicosanoic Acids; D004791:Enzyme Inhibitors; D049990:Membrane Transport Modulators; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D013873:Thionucleotides; D014665:Vasodilator Agents; C400322:cytochrome P-450 CYP2C subfamily; C067410:endothelium-dependent hyperpolarization factor; D012430:Ruthenium Red; D018999:Charybdotoxin; C016957:adenosine-3',5'-cyclic phosphorothioate; D001030:Apamin; D008825:Miconazole; D003577:Cytochrome P-450 Enzyme System; C067407:14,15-episulfide eicosatrienoic acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004851:Epoxide Hydrolases; D015126:8,11,14-Eicosatrienoic Acid; D000218:Adamantane; D001920:Bradykinin