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 2ϩ 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 2ϩ 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 2ϩ influx and prolongs the activation of Ca 2ϩ -dependent K ϩ (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 2ϩ 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. C ytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have been proposed to act as endotheliumderived hyperpolarizing factors (EDHFs) (for review see reference 1). However, the fact that EETs activate iberiotoxinsensitive BK Ca channels 2 while most EDHF-dependent responses are sensitive to the combination of charybdotoxin and apamin that inhibit small (SK Ca ) and intermediate conductance (IK Ca ) K Ca channels, 3 has frequently been used as an argument against the involvement of an EET in the EDHFmediated response. Despite the confusion that exists in the literature related to this topic, it has become clear that probably the most important event in the initiation/generation of EDHF-mediated responses is the rapid hyperpolarization of endothelial cells, an event that precedes the hyperpolarization of vascular smooth muscle cells.The EDHF-mediated relaxation of porcine coronary arteries that is sensitive to the CYP2C9 inhibitor sulfaphenazole and that can be attenuated by antisense oligonucleotides directed against CYP2C; is insensitive to iberiotoxin and sensitive to charybdotoxin and apamin 4 suggesting that the role of EETs in the EDHF phenomenon may not simply be related to the activation of BK Ca channels. There is certainly circumstantial evidence linking EETs with the activation of SK Ca and IK Ca channels inasmuch as the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) attenuates the bradykinin-induced hyperpolarization of native porcine coronary artery endothelial cells...
Webler AC, Michaelis UR, Popp R, Barbosa-Sicard E, Murugan A, Falck JR, Fisslthaler B, Fleming I. Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis.
Objective-Migratory capacity of endothelial progenitor cells (EPCs) and mature endothelial cells (ECs) is a key prerequisite for endothelial repair after denuding injury or endothelial damage. Methods and Results-We demonstrate that caffeine in physiologically relevant concentrations (50 to 100 mol/L) induces migration of human EPCs as well as mature ECs. In patients with coronary artery disease (CAD), caffeinated coffee increased caffeine serum concentration from 2 mol/L to 23 mol/L, coinciding with a significant increase in migratory activity of patient-derived EPCs. Decaffeinated coffee neither affected caffeine serum levels nor migratory capacity of EPCs. Treatment with caffeine for 7 to 10 days in a mouse-model improved endothelial repair after denudation of the carotid artery. The enhancement of reendothelialization by caffeine was significantly reduced in AMPK knockout mice compared to wild-type animals. Transplantation of wild-type and AMPK Ϫ/Ϫ bone marrow into wild-type mice revealed no difference in caffeine challenged reendothelialization. ECs which were depleted of mitochondrial DNA did not migrate when challenged with caffeine, suggesting a potential role for mitochondria in caffeine-dependent migration. Conclusion-These results provide evidence that caffeine enhances endothelial cell migration and reendothelialization in part through an AMPK-dependent mechanism, suggesting a beneficial role for caffeine in endothelial repair.
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