Role of 11,12,15-Trihydroxyeicosatrienoic Acid as a New Endothelium-Derived Relaxing Factor
The endothelium regulates vascular tone through the release of relaxing factors including nitric oxide, prostacyclin, epoxyeicosatrienoic acids and others not yet identified. The purpose of this study was to characterize the 15-lipoxygenase product, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), as a new endothelial relaxing factor. In phenylephrine-constricted rabbit aorta, acetylcholine induced a dose-dependent relaxation in the presence of cyclooxygenase blockade with indomethicin (10 μM) and nitric oxide blockade with nitro-arginine (30 μM) which was eliminated by pretreatment with cinnamyl-3,4,-dihydroxy-a-cyanocinnamate (CDC)(1 μM) a specific lipoxygenase inhibitor, and miconizole (1 μM) a cytochrome P450 inhibitor. Additionally, arachidonic acid (1 nM - 10 μM) relaxed precontracted rabbit aorta, and this relaxation was maximally inhibited by 50% either by increasing the potassium (K+) concentration from 4 mM to 20 mM or by the addition of the K+ channel blockers, apamin (100 nM) plus charybdotoxin (100 nM). In patch-clamp studies of inside-out patches of rabbit aortic smooth muscle, we have identified a 20 pS K+ channel, which is activated by increased calcium concentrations and blocked by apamin (1 μM). Structural analysis of arachidonic acid metabolites from rabbit aorta by gas chromatography/mass spectometry has revealed two 15-lipoxygenase product regioisomers; 11,12,15-THETA and 11,14,15-THETA. The synthesis of these metabolites was inhibited by CDC and miconizole. The addition of the 11,12,15-THETA fraction to precontracted rabbit aorta resulted in a concentration-dependent relaxation, which was blocked by elevated K+. The other regioisomer was without effect. These results suggest that 11,12,15-THETA, a 15-lipoxygenase metabolite, represents a novel endothelium-derived relaxing factor, which induces relaxation possibly through the activation of apamin-sensitive K+ channels located in the vascular smooth muscle.