Cytochrome P450 (CYP)-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. Since many actions of 11,12-EET are dependent on the activation of protein kinase A (PKA), the existence of a cell surface Gs-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 TRPC6V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not 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 while 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 siRNA-mediated downregulation of Gs in endothelial cells had no significant effect on responses stimulated by VEGF or a PKA activator but abolished responses to (±)-11,12-EET. The down regulation of Gq/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 Gs-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET, mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.
- The American Society for Pharmacology and Experimental Therapeutics