Epoxyeicosatrienoic Acids Function as Selective, Endogenous Antagonists of Native Thromboxane Receptors: Identification of a Novel Mechanism of Vasodilation

Abstract

Epoxy- and dihydroxy-eicosatrienoic acids (EETs and DHETs) are vasoactive cytochrome P450 metabolites of arachidonic acid. Interestingly, however, the mechanism(s) by which EETs/DHETs mediate smooth muscle relaxation remains unclear. In contrast to previous reports, where dilation was purportedly large-conductance Ca²⁺-activated K⁺ (BK_Ca) and/or transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel-mediated, 14,15-EET-induced vasodilation [reversal of contractile tone established with the thromboxane receptor (TP) agonist 15-hydroxy-11α,9α-(epoxymethano)prosta-5,13-dienoic acid (U-46619)] was unaltered in BK_Ca and TRPV4 knockout mouse isolated aortae compared with wild-type controls, indicating a significant BK_Ca/TRPV4-resistant mechanism. Whereas all EET and DHET regioisomers reversed U-46619 contraction in rat aortae and mouse mesenteric resistance arteries, these eicosanoids failed to alter phenylephrine-induced contraction, suggesting that they mediated dilation via a “TP-selective” mechanism. Competitive TP antagonism was also observed in nonvascular tissue, including rat fundus and tertiary bronchus, indicating that the effect is not specific to blood vessels. Such effects were TP-selective because 14,15-EET failed to inhibit “non-TP” prostanoid receptor-mediated function in multiple cell/tissue-based assays (K_b > 10 μM). In accordance, 14,15-EET inhibited specific [³H]7-(3-((2-((phenylamino)carbonyl)hydrazino)-methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ-29548) binding to human recombinant TP receptor, with a K_i value of 3.2 μM, and it showed weaker affinity for non-TP prostanoid receptors, including DP, FP, EP_1–4, and IP receptors (K_i values of 6.1, 5.3, 42.6, 19.7, 13.2, 20.2, and >25 μM, respectively) and no appreciable affinity (K_i values >10 μM) for a diverse array of pharmacologically distinct receptors, including the leukotriene receptors Cys-LT_1/2 and BLT₁. As such, EETs/DHETs represent a unique class of “endogenous” G protein-coupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Thus, EETs/DHETs represent novel autoregulatory agents, directly modulating the actions of cyclooxygenase-derived eicosanoids following arachidonic acid mobilization.