Renal vasodilator activity of 5,6-epoxyeicosatrienoic acid depends upon conversion by cyclooxygenase and release of prostaglandins

The 5,6-epoxyeicosatrienoic acid (5,6-EET), a renal vasodilator metabolite of arachidonic acid via cytochrome P450 (P450) requires cyclooxygenase for expression of its vasoactivity as the responses are inhibited by indomethacin and other aspirin-like drugs. We now report on the metabolism of 5,6-EET by rabbit kidneys in order to characterize those metabolites that may account for its vasoactivity. The 5,6-EET was injected close-arterially into the rabbit isolated Krebs-Henseleit perfused kidney, preconstricted with phenylephrine, and the effluent collected throughout the response period. Basal collections, following injection of 100 μl of vehicle, were made at 20-min intervals before each 5,6-EET injection. Prior to acidic extraction, deuterated 6-keto-prostaglandin (PG) F^1α and PGE₂ were added as internal standards. The extracts were separated by TLC and prostaglandins were derivatized for gas chromatography-mass spectrometry analysis using a negative ion chemical ionization mode. Injection of 0.5, 1, 5, 10, and 20 μg of 5,6-EET (n = 4) resulted in dose-related decreases in perfusion pressure of 6 ± 2, 12 ± 4, 21 ± 4, 26 ± 4, and 27 ± 7 mm Hg, respectively. Basal perfusates contained 6-keto-PGF_1α and PGE₂, levels of which were increased by 2-fold or more by 5,6-EET. Perfusates, collected during 5,6-EET administration, also contained 5-hydroxy-PGI₁ and 5,6-epoxy-PGE₁, cyclooxygenase metabolites of 5,6-EET. This is the first report of the recovery and identification of these 5,6-EET metabolites from an intact organ. Since the responses to 5,6-EET are endothelial-dependent, we also studied the profile of eicosanoids formed following incubation of 5,6-EET with cultured bovine pulmonary endothelial cells. Endothelial cells metabolized 5,6-EET to products with a similar radioactive profile on reverse-phase high pressure liquid chromatography compared to kidney perfusates. We compared the vasodilator activity of 5,6-epoxy-PGE₁ and 5-hydroxy-PGI₁, chemically synthesized by us from PGE₂ and PGF_2α, respectively, with PGE₂ and PGI₂ in the rabbit kidney. The 5,6-epoxy-PGE₁ was equipotent to PGE₂ as a vasodilator. The ED₅₀ values for 5,6-EET, 5,6-epoxy-PGE₁, and PGE₂ were 4.69, 0.43, and 0.42 nmol, respectively. Although PGI₂ was a potent vasodilator (ED₅₀, 0.24 nmol), 5-hydroxy-PGI₁ was devoid of activity. Thus, the cyclooxygenase-dependent vasoactivity of 5,6-EET in the rabbit kidney has two components: release of vasodilator prostaglandins, PGE₂ and PGI₂, and metabolism of 5,6-EET to a prostaglandin analog, 5,6-epoxy-PGE₁.