Role of cytochrome P450 (CYP) metabolites of arachidonic acid in the regulation of cAMP in HEK293 cells

Abstract

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which in turn are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). EETs are known to modulate a number of vascular and renal functions but the exact signaling mechanism(s) of these EET-mediated effects remains unknown. Purpose: To investigate the role of EETs and DHETs in regulating cAMP production via adenylyl cyclase (AC) in a human embryonic kidney cell line (HEK 293). Method: HEK 293 cells were treated with vehicle, forskolin, epinephrine, 11,12-EET, 11,12-DHET, as well as potential pathway and G-protein inhibitors to assess changes in cAMP production. Results: Co-administering 11,12-EET with forskolin or epinephrine effectively eliminated the increased cAMP levels observed in cells treated with forskolin alone. The inhibitory effect of EETs on forskolin-mediated cAMP production was abolished when cells were treated with a sEH inhibitor (tAUCB). 11,12-DHET also negated the effects of forskolin, suggesting the inhibitory effect observed in EET-treated cells could be attributed to the downstream metabolites, DHETs. In contrast, inhibition of phosphodiesterase IV (PDE4) with rolipram eliminated the effects of EETs or DHETs, and Gαi with pertussis toxin also resulted in enhanced cAMP production. Conclusion: Our data suggest that DHETs regulate cAMP production via PDE4 and Gαi protein. Moreover, they provide novel evidence as to how EET-mediated signaling may alter G-protein coupling in HEK 293 cells