Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin Submitted 6 July 2004 ; accepted in final form 15 September 2004 We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10 –9 –10 –4 M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 ± 0.5% and EC 50 of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 µM), NS-398 (10 µM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 µM), nordihydroguaiaretic acid (NDGA; 1 µM), and ebselen (1 µM). HPLC analysis showed that [ 14 C]AA was metabolized by mesenteric arteries to PGI 2 , PGE 2 , THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI 2 and PGE 2 was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PGI 2 , PGE 2 , and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K + (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways. endothelium-derived factors; trihydroxyeicosatrienoic acid; prostaglandin E 2 ; prostaglandin I 2 ; endothelium-derived hyperpolarizing factor Address for reprint requests and other correspondence: W. B. Campbell, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: wbcamp{at}mcw.edu )