Epoxyeicosatrienoic Acids Regulate Trp Channel–Dependent Ca 2+ Signaling and Hyperpolarization in Endothelial Cells

Abstract

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. Methods and 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.