Submitted on November 12, 2007
Accepted on January 11, 2008
Identification of 15-Hydroxy-11,12-Epoxyeicosatrienoic Acid as a Vasoactive 15-Lipoxygenase Metabolite in Rabbit Aorta
Yuttana Chawengsub1, Nitin Aggarwal2, Kasem Nithipatikom1, Kathryn M Gauthier3, Siddam Anjaiah4, Bruce D Hammock, John R. Falck5, and William B. Campbell6*
1 Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
2 Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3 Dept of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
4 UT Health Science Center, Memphis, Tennessee, United States
5 Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, Texas, United States
6 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee,, Wisconsin, United States
* To whom correspondence should be addressed. E-mail: wbcamp{at}mcw.edu.
Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-LO-I metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We proposed that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. Following incubation of aorta with 14C-AA, metabolites were extracted, and the HEETAs resolved by HPLC. Mass spectrometric analyses identified 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid sensitive 15-H-11,12-EETA as methoxy-dihydroxy-eicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) (10-6 M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus, 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57 kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10-7-10-4 M) and acetylcholine (10-9-10-6 M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K+] from 4.8 mM to 20 mM. AA (3x10-6 M) induced cell membrane hyperpolarization (from -31.0 ± 1 mV to -46.8 ± 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA and mediates relaxations by membrane hyperpolarization, 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.
