title: Regulation of forskolin-induced cAMP production by cytochrome P450 epoxygenase metabolites of arachidonic acid in HEK293 cells.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  The purpose of this study is to investigate the role of EETs and DHETs in regulating cyclic adenosine monophosphate (cAMP) production via adenylyl cyclase in a human embryonic kidney cell line (HEK293).
  
  
  METHODS
  HEK293 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 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 that 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 inhibition of Gαi with pertussis toxin also resulted in enhanced cAMP production.
  
  
  CONCLUSIONS
  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 HEK293 cells.
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, T6G 2N8, Canada.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Abukhashim
authors.forename: Glenis J
authors.initials: GJ
authors.lastname: Wiebe
authors.forename: John M
authors.initials: JM
authors.lastname: Seubert
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis*; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D013997:Time Factors
chemical_list: C410128:11,12-dihydroxyeicosatrienoic acid; C494022:14,15-eicosa-5-enoic acid; D005576:Colforsin; D016718:Arachidonic Acid; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D000090762:Cytochrome P-450 CYP2J2; D015126:8,11,14-Eicosatrienoic Acid