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

Siangjong L, Gauthier KM, Pfister SL, Smyth EM, Campbell WB. Endothelial 12(S)-HETE vasorelaxation is mediated by thromboxane receptor inhibition in mouse mesenteric arteries. Am J Physiol Heart Circ Physiol 304: H382-H392, 2013. First published November 30, 2012; doi: 10.1152/ajpheart.00690.2012.-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 A(2)-, PGF(2 alpha)-, and 8-iso PGF(2 alpha)-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 mu M) inhibited constrictions to U46619 but not phenylephrine. To investigate the role of thromboxane A(2) (TP) receptors in 12-HETE vascular actions, [H-3]SQ29548 radioligand binding studies were performed in mouse platelets. U46619, 12(R)-HETE, and 12(S)HETE displaced [3H] SQ29548 binding with IC(50)s of 0.07, 0.32, and 1.73 mu M, respectively. Both 12(S)- and 12(R)-HETE inhibited intracellular calcium increases induced by U46619 (10 nM) in HEK293 cells overexpressing TP alpha receptor (65.5% and 45.1%, respectively) and coexpressing prostacyclin (IP) and TP alpha receptors (58.0% and 27.1%, respectively). The LO inhibitor NDGA (10 mu M) 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.