Role of Arachidonic Acid Lipoxygenase Metabolites 1 in Acetylcholine Relaxations of Mouse Arteries

39 Arachidonic acid (AA) metabolites function as endothelium-derived hyperpolarizing 40 factors (EDHFs) in arteries of many species. They mediate cyclooxygenase (COX)and nitric 41 oxide (NO)-independent relaxations to acetylcholine. However, the role of AA metabolites as 42 relaxing factors in mouse arteries remains incompletely defined. Acetylcholine caused 43 concentration-dependent relaxations of mouse thoracic and abdominal aorta, carotid, femoral and 44 mesentery arteries (maximal relaxation = 57±4%, 72±4%, 82±3%, 80±3% and 85±3%, 45 respectively). The NO synthase inhibitor, nitro-l-arginine (L-NA, 30 μM) blocked relaxations in 46 thoracic aorta and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in 47 abdominal aorta, carotid, femoral and mesenteric arteries (maximal relaxation = 31±10%, 48 33±5%, 41±8% and 73±3%, respectively). In mesenteric arteries, NOand COX-independent 49 relaxations to acetylcholine were inhibited by the lipoxygenase (LO) inhibitors NDGA (10 μM) 50 and BW755C (200 μM), the potassium channel inhibitor, apamin (1 μM) and 60 mM KCl and 51 eliminated by endothelium removal. They were not altered by the cytochrome P450 inhibitor, 52 MS-PPOH (20 μM) or the epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 μM). AA 53 relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse phase54 HPLC analysis revealed arterial [C]-AA metabolites that co-migrated with prostaglandins 55 (PG), trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs) 56 and hydroxyeicosatetraenoic acids (HETEs). EETs were not observed. Mass spectrometry 57 confirmed the identity of 6-keto PGF1α, PGE2, 12and 15-HETE, HEETAs and 11,12,15and 58 11,14,15-THETAs. AA metabolism was blocked by NDGA and endothelium removal. 59 11(R),12(S),15(S)-THETA relaxations (maximal = 73±3%) were endothelium independent and 60 blocked by 60 mM KCl. Western immunoblot and RT-PCR of aorta and mesenteric arteries 61