The biological actions of 11,12-epoxyeicosatrienoic acid in endothelial cells are specific to the R/S-enantiomer and require the G(s) protein.
Cytochrome P450-derived epoxides of arachidonic acid [i.e., the epoxyeicosatrienoic acids (EETs)] are important lipid signaling molecules involved in the regulation of vascular tone and angiogenesis. Because many actions of 11,12-cis-epoxyeicosatrienoic acid (EET) are dependent on the activation of protein kinase A (PKA), the existence of a cell-surface G(s)-coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12-EET are enantiomer specific and linked to a potential G protein-coupled receptor, we assessed 11,12-EET-induced, PKA-dependent translocation of transient receptor potential (TRP) C6 channels, as well as angiogenesis. In primary cultures of human endothelial cells, (±)-11,12-EET led to the rapid (30 seconds) translocation a TRPC6-V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not by 11(S),12(R)-EET or (±)-14,15-EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)-11,12-EET and 11(R),12(S)-EET, whereas 11(S),12(R)-EET and 11,12-dihydroxyeicosatrienoic acid were without effect. The effects of (±)-11,12-EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists, and TRP channel trafficking was also prevented by a PKA inhibitor. The small interfering RNA-mediated downregulation of G(s) in endothelial cells had no significant effect on responses stimulated by vascular endothelial growth or a PKA activator but abolished responses to (±)-11,12-EET. The downregulation of G(q)/11 failed to prevent 11,12-EET-induced TRPC6 channel translocation or the formation of capillary-like structures. Taken together, our results suggest that a G(s)-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET and mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.).
Yindi
Y
Ding
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.).
Timo
T
Frömel
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.).
Rüdiger
R
Popp
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.).
John R
JR
Falck
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.).
Wolf-Hagen
WH
Schunck
Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University Frankfurt am Main, Germany (Y.D., T.F., R.P., I.F.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Max-Delbrück Center for Molecular Medicine, Berlin, Germany (W.-H.S.) fleming@em.uni-frankfurt.de.
Ingrid
I
Fleming
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000494:pharmacology; D013237:Stereoisomerism; D050052:TRPC Cation Channels / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
C410128:11,12-dihydroxyeicosatrienoic acid; D043925:Angiogenesis Inducing Agents; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D042461:Vascular Endothelial Growth Factor A; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D017868:Cyclic AMP-Dependent Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs; D015126:8,11,14-Eicosatrienoic Acid