Question:

Cytochrome P450 (CYP)-derived epoxides of arachidonic acid i.e. the epoxyeicosatrienoic acids (EETs) are important lipid signalling molecules implicated in the regulation of vascular tone and the attenuation of inflammation. The molecular events linking the lipid with cellular signalling are unclear but as many of the actions of 11,12-EET are dependent on the activation of protein kinase (PK) A, the existence of a cell surface Gαs-protein-coupled receptor has been postulated. We used the 11,12-EET-induced, PKA-dependent translocation of TRPC6 channels to screen for the existence of an EET receptor in endothelial cells.

Methods:

TRPC6 was overexpressed in primary human umbilical vein endothelial cells (HUVEC) or a related cell line (HUV-EC-c) and TRPC6 translocation to the plasma membrane following EET-stimulation was assessed by confocal microscopy. siRNA was used to downregulate Gαs expression.

Results:

Stimulating HUVEC or HUV-EC-c with 11,12- EET led to the translocation of TRPC6 channels from the Golgi apparatus to caveolae. This response was sensitive to the EET-antagonists 14,15-EEZE and miconazole as well as the cAMP analogue Rp-cAMPs. 14,15-EET did not elicit translocation. In HUV-EC-c cells siRNA directed against Gαs abolished the 11,12-EET-induced translocation of TRPC6 but was without effect in cells stimulated with forskolin or Sp-cAMPs.

Conclusions:

Taken together, our results suggest that a Gαs-coupled receptor in the endothelial cell membrane responds to 11,12-EET to mediate the PKA-dependent translocation and activation of TRPC6 channels.