Characterization of stable epoxyeicosatrienoic acid agonists: Use for study of signaling mechanisms.

摘要

Epoxyeicosatrienoic acids (EETs), 5,6-, 8,9-, 11,12- and 14,15-EETs, are cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid. In the coronary circulation, EETs cause potent vasodilation by activation of large conductance, Ca2+-activated K+ (BK Ca) channels and represent endothelium-derived hyperpolarizing factors (EDHFs). EETs also regulate vascular homeostasis through numerous signaling pathways. The initiation site of EET signaling, however, remains unclear. Many studies support a plasma membrane EET receptor as well as an intracellular EET-binding protein such as peroxisome proliferator-activated receptor gamma. However, active enzymatic metabolism of EETs has limited the study of EET signaling. 5,6-EET, unlike other isomers, undergoes spontaneous hydrolysis in aqueous solutions. 5,6-EET is also a substrate for cyclooxygenase (COX). The role of COX metabolism in 5,6-EET-mediated bovine coronary artery (BCA) relaxation has not been determined.;The goal of the current studies was to develop stable EET agonists to examine EET signaling mechanisms. These agonists include (1) hydrolysis-resistant 5,6-EET agonists, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid ( PTPA), 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPAs), (2) a series of 14,15-EET-sulfonamides, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA ) and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA ), in which the 14,15-EET moiety was tagged with a radioiodinable phenyl ring, a biotin, or a photoreactive group, respectively, and (3) 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), in which an iodine substitutes a hydrogen at the C20 methyl.;PTPA caused BCA relaxation with equal potency and activity as 5,6-EET. PTPA relaxation was inhibited by a COX inhibitor, an ATP-sensitive K + (KATP) channel blocker, and a BKCa channel blocker. The effects of COX inhibition and KATP blockade were redundant whereas the effects of BKCa blockade and COX/KATP inhibition were additive. PDPAs resisted COX metabolism. BCA relaxation to PDPAs were 50% less than PTPA and 5,6-EET. PDPA relaxations were sensitive to BK Ca blockade but not to COX/KATP inhibition. These studies suggested that PTPA induces relaxation through dual COX/EDHF pathways and PDPAs represent the EDHF component of 5,6-EET vasoactivity.;14,15-EET-PISA, 14,15-EET-BSA and 14,15-EET-BZDC-SA induced BCA relaxation with equal potency and activity as 14,15-EET. The relaxations were blocked by high extracellular K+, BKCa blockade and an EET antagonist, 14,15-epoxyeicosa-5(Z)-enoyl-methylsulfonamide (14,15-EE5ZE-mSA). 14,15-EET-PISA was metabolized to 14,15-dihydroxyeicosatrienoyl-PISA (14,15-DHET-PISA) by soluble epoxide hydrolase in BCA and U937 cells but not U937 cell membrane fractions. 14,15-EET-P125ISA bound to human U937 membranes in a time-dependent, concentration-dependent and saturable manner with estimated KD and Bmax of 148.3 +/- 36.4 nM and 3.3 +/- 0.5 pmol/mg protein, respectively. Therefore, 14,15-EET-sulfonamides are full 14,15-EET agonists and 14,15-EET-P125ISA is a new radiolabeled tool to study EET metabolism and binding.;20-1-14,15-EE8ZE stimulated U937 cell cAMP production with similar potency but 50% efficacy as 11,12-EET. It also relaxed BCA with equal potency and activity as EETs. Both agonist activities were blocked by the EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE). 20-125I-14,15-EE8ZE binding of U937 membranes was performed and a reversible specific EET-binding site was identified. The binding was saturable with a KD and Bmax of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively, and was reversed by 11,12-EET (20 muM). The non-hydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS, 500 nM) reduced the Bmax by 45%. Eicosanoids competed for 20-125I-14,15-EE8ZE binding in an order of 11,12-EET > 14,15-EE5ZE ≈ 14,15-EET 15-hydroxyeicosatetraenoic acid (15-HETE) > 14,15-EET-thiirane > 14,15-DHET. This order was in agreement with the efficacy of cAMP activation. These data suggest that EET-binding of U937 membranes occurs with high affinity, high abundance and is sensitive to GTPgammaS modulation. This binding site could represent a G-protein coupled receptor for EETs.;14,15-EET-P125ISA, 20-125I-14,15-EE8ZE and its methyl ester 20-125I-14,15-EE8ZE-ME were incubated with membrane proteins from bovine hearts, bovine coronary arteries, U937 cells, rat and bovine myocardial mitochondria. After resolving the proteins on SDS-PAGE gels, a 48kD band that co-distributed with mitochondria was specifically radiolabeled and detected on autoradiography. The radiolabeling was reversed by high SDS treatment and selectively inhibited by four EET isomers and 14,15-EE5ZE. Mass spectrometric analysis of the 48kD band identified F1 ATPase beta subunit as a candidate EET-binding protein. In vitro studies showed that 11,12- and 14,15-EET inhibited mitochondrial F1 ATP hydrolase and ATP synthase activities.;In conclusion, this thesis provides evidence supporting the presence of a G-protein coupled EET-receptor, a new mitochondrial action of EETs and a COX/KATP dependent mechanism of 5,6-EET mediated BCA relaxation. Importantly, the EET analogs characterized by these studies provide pharmacological tools for the determination of EET signaling mechanisms.