11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid:: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15( S)-THETA. Of the eight isomers, only 11( R), 12( S), 15( S)trihydroxyeicosa-5(Z),8(Z),13(E)- trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7) - 10(-4) M) relaxed the rabbit aorta in a concentration- related manner ( maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+- activated K+ channels. In comparison, 11(S), 12(R), 15(S), 5(Z), 8(Z), 13( E)- THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R), 12(S), 15(S), 5(Z), 8(Z), 13( E)- THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)trihydroxyeicosa- 5( Z), 8( Z), 13( E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.