Endothelial 12(S)-HETE vasorelaxation is mediated by thromboxane receptor inhibition in mouse mesenteric arteries

Abstract

Arachidonic acid (AA) metabolites mediate endothelium-dependent relaxation in many vascular beds. Previously, we identified the major AA 12/15-lipoxygenase (12/15-LO) metabolite of mouse arteries as 12-hydroxyeicosatetraenoic acid (12-HETE). The goal was to determine the stereospecific configuration of mouse vascular 12-HETE and characterize the role of 12-HETE stereoisomers in the regulation of vascular tone. Using normal, reverse phase, and chiral HPLC, the stereospecific configuration was identified as 12( S)-HETE. 12( S)-HETE relaxed U46619-, carbocyclic thromboxane A2-, PGF2α-, and 8-iso PGF2α-preconstricted mesenteric arteries, but not phenylephrine-preconstricted arteries. 12( R)-HETE was more potent than 12( S)-HETE in relaxing U46619-preconstricted mouse arteries (maximum relaxations = 91.4 ± 2.7% and 71.8 ± 5.9%, respectively). Neither 12-HETE isomer caused constriction. Pretreatment with 12( S)- or 12( R)-HETE (1 μM) inhibited constrictions to U46619 but not phenylephrine. To investigate the role of thromboxane A2(TP) receptors in 12-HETE vascular actions, [3H]SQ29548 radioligand binding studies were performed in mouse platelets. U46619, 12( R)-HETE, and 12( S)-HETE displaced [3H]SQ29548 binding with IC50s of 0.07, 0.32, and 1.73 μM, respectively. Both 12( S)- and 12( R)-HETE inhibited intracellular calcium increases induced by U46619 (10 nM) in HEK293 cells overexpressing TPαreceptor (65.5% and 45.1%, respectively) and coexpressing prostacyclin (IP) and TPαreceptors (58.0% and 27.1%, respectively). The LO inhibitor NDGA (10 μM) reduced AA relaxations in arteries preconstricted with U46619 but not phenylephrine. These results indicate that exogenous and endogenous 12( S)-HETE relax mouse mesenteric arteries that are preconstricted with thromboxane agonists. These 12( S)-HETE relaxations are mediated by TP receptor competitive inhibition and inhibition of TP agonist-induced increases in intracellular calcium.