Abstract 1300: Identification of a Novel Vasodilator in Rabbit Aorta: 15-Hydroxy-11,12-Epoxyeicostrienoic Acid, a 15-Lipoxygenase Metabolite of Arachidonic Acid
Arachidonic acid causes endothelium-dependent relaxation of rabbit aorta that is blocked by lipoxygenase inhibitors. We previously demonstrated that a 15-lipoxygenase metabolite of arachidonic acid, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) contributes to arachidonic acid and acetylcholine relaxations. In this study, we assessed the hypothesis that in rabbit aorta, arachidonic acid is metabolized by 15-lipoxygenase to an unstable precursor of 11,12,15-THETA, namely 15-hydroxy-11,12-epoxyeicostrienoic acid (15-H-11,12-EETA). Following the incubation of aorta with arachidonic acid, metabolites were resolved by reverse-phase HPLC. Analysis by mass spectrometry identified a vasoactive metabolite as 15-H-11,12-EETA. Treatment of aortic incubations with water and acid yielded 11,12,15-THETA (353 m/z, M-H ion), while treatment with methanol and acid yielded methoxy-dihydroxy-eicosatrienoic acid (367 m/z, M-H ion), indicating that 15-H-11,12-EETA is an acid sensitive precursor of 11,12,15-THETA. Hydrolysis of 15-H-11,12-EETA to methoxy-dihydroxy-eicosatrienoic acid in acid and methanol is a means to evaluate 15-H-11,12-EETA produced. Western immunoblot of rabbit aortic homogenates revealed a 62-kDa band corresponding to soluble epoxide hydrolase. The soluble epoxide hydrolase inhibitor, 1-amantidyl-3-dodecyl-ureic acid (ADUA) (1 βM) increased formation of methoxy-dihydroxy-eicosatrienoic acid by aorta, indicating soluble epoxide hydrolase can convert 15-H-11,12-EETA to 11,12,15-THETA. In preconstricted aortic rings, arachidonic acid (10−7-10−4 M) caused concentration-related relaxations (maximum relaxation ± 10±2%) that were enhanced (maximum relaxation ± 22±2%) by pretreatment with the ADUA (1 μM). In conclusion, this study demonstrates that 15-H-11,12-EETA is produced in rabbit aorta, is hydrolyzed by soluble epoxide hydrolase to 11,12,15-THETA, and causes vascular relaxation with higher efficacy than 11,12,15-THETA. These findings suggest that 15-H-11,12-EETA represents a vasodilator metabolite of 15-lipoxygenase in rabbit and may have an important role in regulating vascular tone.